HIV transgenic animals and uses therefor

The invention provides transgenic animals comprising a lentiviral transgene, such as an HIV transgene. Also within the scope of the invention are cells and eggs from the transgenic animal. Further included are methods for identifying therapeutic compounds for preventing lentiviral infection and treating associated disease (e.g. AIDS).

BACKGROUND OF THE INVENTION 
Human immunodeficiency virus (HIV) is an etiological agent of Acquired 
Immune Deficiency Syndrome (AIDS). AIDS was first reported in the United 
States of America in 1981. As of January 1997, approximately 1.5 million 
cases of AIDS in adults and children had been reported to the World Health 
Organization (WHO); however, because reporting is difficult, WHO estimates 
that there were more than 8.4 million cases, about 580,000 of which reside 
in the United States. However, the number of HIV infected individuals is 
much higher: as of January 1997, WHO estimated that there were 
approximately 29.4 million HIV infected individuals world-wide, with about 
1 million in the United States. It has been estimated that by the year 
2000, between 40 and 100 million individuals will be infected with HIV. 
HIV, which has also been referred to as lymphadenopathy-associated virus 
(LAV), HTLV-III, or AIDS related virus (ARV), is a lentivirus from the 
family of retroviruses and is composed of RNA consisting of about 9,700 
base pairs, three gag proteins (having molecular weights of 55,000, 24,000 
and 17,000 daltons), a reverse transcriptase (molecular weights of 66,000 
and 51,000 daltons have been detected), three glycoproteins (two molecules 
having molecular weights of 120,000 and 41,000 daltons, and their 
precursor, a molecule with a molecular weight of 160,000 daltons, 
hereinafter abbreviated as gp120, gp41 and gp160, respectively) which 
comprise the envelope, and other components. Exposed, envelope proteins 
are particularly important for viral infection and therefor also, the 
prevention thereof. As a result of proteolysis, gp1 60 is cleaved into 
gp120 and gp41. Gp4l is a transmembrane protein which is incorporated into 
the lipid bilayer of the viral envelope, while gp120 is exposed on the 
outside of the envelope and some of it is released from the virus. Both 
gp4l and gp120 possess many sugar-binding sites, and about half of the 
gp120 molecule is comprised of sugars. The gp120 molecule binds to the CD4 
antigens on the surface of cells, in particular helper T cells. Once HIV 
is bound to CD4 via gp 120, another env gene product, gp41, mediates 
fusion between the membranes of the cell and the virus allowing the core 
of the virus to enter the cell. Gp120, which is expressed on the plasma 
membrane of infected cells before virus is released, can bind to CD4 on 
another cell, initiating a membrane fusion event resulting in syncytia 
formation, and HIV genomes can be passed between the fused cells directly. 
The env gene (gp120) is the primary determinant of cell tropism for both 
HIV and Simian Immunodeficiency Virus (SIV). Variable region 3 (V3) of 
gp120 is a key component within env that determines cell tropism. The 
efficiency of replication and the ability to induce the syncytia formation 
are also affected by changes in the V3 loop. 
The first HIV virus isolated is referred to as HIV-1 and is generally 
described in several articles, e.g., Barre-Sinoussi et al., Science 
220:868, 1983; Gallo et al., Science 224:500, 1984; Popovic et al., 
Science 224:497, 1984; and Levy et al., Science 225:840, 1984, each of 
which is hereby incorporated by reference. Various isolates of HIV-1 have 
been obtained from North America, Western Europe and Central Africa. These 
isolates differ somewhat in their nucleotide sequence, but the proteins 
they encode are generally antigenically cross-reactive. 
A second virus related to HIV-1 has been isolated and termed HIV-2 (Guyader 
et al., Nature 326:662, 1987; Brun-Vezinet et al., The Lancet 1:128, 1987; 
and Clavel et al., Science 233:343, 1986). The genetic organization of 
HIV-2 is similar to that of HIV-1. Of the two distinct subtypes, HIV-1 is 
predominant and found throughout the world, whereas HIV-2 has been 
isolated primarily in West African countries such as Guinea Bissay, Ivory 
Coast, and Senegal, whith some cases also identified in the Americas and 
western Europe. Epidemiological studies suggest that the incubation period 
for HIV-2 for the development of disease is longer than for HIV-1. 
HIV isolates from around the world were found to differ in nucleotide 
sequence. These sequences have been collected in a specialized database 
(Myers et al. (1994) Los Alamos National Laboratory, Los Alamos, N.Mex.). 
Two major groups of HIV has been identified. Viruses of group M (for 
"main") are responsible for the majority of infections worldwide; group O 
(for "outgroup") is a relatively rare group currently found in Cameroon, 
Gabon, and France. Group M can be divided into at least eight distinct 
subtypes or clades (A through H) (Myers, supra; Louwagie et al. (1995) J. 
Virol. 69:263). Isolates from HIV-1 from different clades may differ by 
30-40% in the amino acid sequence of the gp120 SU protein; isolates within 
a lade vary from 5% to 20%. Clade B predominates in North America and 
Europe and lade E predominates in northern Thailand. Similarly, there are 
five HIV-2 sequence subtypes. 
A group of viruses isolated from monkeys, termed simian immunodeficiency 
virus (SIV or STLV-III), is related to HIV-1 and HIV-2, particularly the 
latter. See Daniel et al., Science 228:1201-1204 (1985); Kanki et al., 
Science 230:951-954 (1985); Chakrabarti et al., Nature 328:543-547 (1987); 
and Ohta et al., Int'l. J. Cancer 41:115-222 (1988). Members of this viral 
group exhibit minor variations in their genomic sequences, and have some 
differences in their restriction enzyme maps. 
Although human CD4 is essential for HIV infection, it is not sufficient. 
Expression of human CD4 on rodent cells renders them capable of binding 
virus but still nonpermissive for fusion or infection (Maddon et al. 
(1986) Cell 47:333). The host component or coreceptors, sometimes referred 
to as the "fusion receptors", were identified only recently. These 
coreceptors are receptors for chemokines (i.e. small proteins which serve 
as chemoattractants in inflammation) and they permit HIV infection of 
virtually any mammalian or avian cell that expresses human CD4 (Bates 
(1996) Cell 86:1-3). The most important coreceptors are CXCR4 (also called 
"fusin" or "LESTR) (Endres et al. (1996) Cell 87:745; Feng et al. (1996) 
Science 272:872) and CCR5 (Akhatib et al. (1996) Science 272:1955; Choe et 
al. (1996) Cell 85:1135; Deng et al. (1996) Nature 381:661; Doranz et al. 
(1996) Cell 85:1149; and Dragic et al. (1996) Nature 381:667). CXCR4 is 
the receptor for the chemokine SDF-1, whereas CCR5 serves as a receptor 
for the chemokines MIP-1.alpha. and .beta. and RANTES. These coreceptors 
play a crucial function for viral entry into cells, and they are also the 
principal determinants of tropism among CD4+ cells. 
Two distinct types of HIV-1 have been identified based on the cells in 
which they replicate in vitro. Viruses that replicate in T cell lines, but 
not macrophages or monocytes, are referred to as T tropic, whereas viruses 
with the complementary specificity are referred to as M tropic. The 
tropism is at least a function of the coreceptor: M tropic viruses can use 
only CCR5 for entry, and T tropic viruses use CXCR4. A few dual tropic 
isolates capable of using both are also known. T tropic viruses often 
cause infected cells to fuse with uninfected cells if the latter express 
both human CD4 and CXCR4. Such viruses are referred to as 
'syncytium-inducing" (SI). All isolates can infect activated T cells 
freshly isolated from peripheral blood, which are present in PBMC 
cultures, since such cells express both CXCR4 and CCR5. Furthermore, cell 
tropisms are not fixed and can change when the virus is passaged in cell 
culture (Metlzer et al. (1990) Immunology Today 11:217; Levy (1993) 
Microbiol. Rev. 57:183). 
Two animal species (i.e., man and chimpanzee) are known to be susceptible 
to HIV infection, but only in man does the disease develop. HIV-1 
transgenic mice carrying intact copies of the HIV-1 provirus have been 
obtained (Leonard et al. (1988) Science 242:1665). These mice develop a 
spontaneous and fatal disease that mimics some of the features described 
in human AIDS. Other HIV-1 transgenic mice carrying the HIV-1 proviral DNA 
in which deletions have been introduced have also been produced (see, 
e.g., Dickie et al. (1991) Virology 185:109; Santoro et al. (1994) Virol. 
201:147). 
However, none of these transgenic mice closely model the development of 
AIDS in humans. In particular, none of the HIV transgenic mice express 
gp120 on the surface of their T cells. Thus, syncytium formation between 
HIV infected cells and CD4+ cells, e.g., T cells, which is reported to 
occur in humans and which is in fact the mechanism by which HIV is 
transmitted from one cell to another without the production of infectious 
HIV particles, does not occur in HIV transgenic mice. In addition, since 
HIV transgenic mice do not express gp120 on the surface of infected cells 
and all of the neutralizing antibodies in humans have mapped to the 
envelope protein, gp160, or one of its component parts (gp120 or gp41), 
transgenic, HIV mice are not particularly useful for developing human HIV 
vaccines. 
Thus, there is a need for animal models of AIDS and other lentiviral 
diseases, which more closely model infection and disease progression as it 
occurs in humans. 
SUMMARY OF THE INVENTION 
In one aspect, the present invention features non-human animal models of 
lentiviral (e.g., HIV) infection and development of disease (e.g. AIDS). 
Preferred non-human animals comprised of a lentiviral transgene are larger 
than a mouse. Other preferred non-human, transgenic animals are smaller 
than a monkey. A particularly preferred non-human, transgenic animal is a 
transgenic rat. 
In preferred embodiments, the transgene comprises essentially all of a 
viral genome, i.e., the transgene comprises at least about 70%, at least 
about 80%, at least about 90% or at least about 95% of a wild-type viral 
genome. Also within the scope of the invention are transgenic animals in 
which the transgene comprises a smaller portion of the wildtype virus, 
(e.g. less than about 70% of the viral genome). For example, the 
transgenic animal can comprise a transgene encoding a single protein. 
In a preferred embodiment, the transgenic, non-human animal is comprised of 
an HIV transgene. Exemplary HIV proteins for inclusion in the transgene 
include an envelope protein (e.g., gp120 and gp40), a reverse 
transcriptase, a protease, an integrase, a ribonuclease, a nucleocapsid 
core factor (gag), a transcriptional activator (e.g., tat and vpr) or 
proteins encoded by the genes vif, vpu, and nef 
In another preferred embodiment, the transgenic, non-human animal expresses 
the HIV proteins. In a particularly preferred embodiment, the animal 
expresses the HIV protein gp120 on the surface of its peripheral blood 
mononuclear cells (PBMCs). In a further preferred embodiment, the 
transgenic, non-human animal expresses at least one HIV coreceptor (e.g. 
CCR5 or CXCR4). 
In another embodiment, in addition to the lentiviral transgene, the 
transgenic, non-human animal is comprised of at least one additional 
transgene. In a preferred embodiment, the additional transgene is a human 
CD4 receptor gene. Expression by the animal of both HIV and human CD4 
allows the HIV particles produced to enter the CD4 expressing cells, thus 
resulting in HIV infection. In another preferred embodiment, the 
additional transgene is an HIV coreceptor, such as CCR5 or CXCR4, which 
further aids in HIV infection. In a further preferred embodiment, the 
additional transgene is a gene involved in a disease or condition that is 
associated with AIDS (e.g. hypertension, Kaposi's sarcoma, cachexia, 
etc.). 
In another preferred embodiment, the non-human animal containing an HIV 
transgene exhibits at least one symptom or phenotype characteristic of 
human HIV infection and/or development of AIDS (e.g. development of 
cataracts, cachexia or lesions (e.g. skin lesions, for example, resulting 
from psoratic dermatitis, hyperkerstotic lesions, kidney sclerotic lesions 
or inflammatory lesions of the central nervous system). 
In another aspect, the invention features methods for producing the 
transgenic animals described herein. A preferred method comprises the 
steps of: (a) obtaining a non-human animal egg containing a lentivirus 
transgene; (b) implanting the egg of step (a) into a female non-human 
animal; (c) selecting offspring containing the transgene to thereby obtain 
a founder animal; and (d) crossing the founder animal with another animal 
preferably of the same species, but opposite sex, to thereby produce a 
transgenic animal comprising a lentivirus transgene. 
In one embodiment, the lentiviral transgene, in step (a), is an HIV 
transgene. In a preferred embodiment, the HIV transgene is infectious. For 
example, the transgene can be supplied by a wild-type HIV provirus, e.g, 
an HIV-1 or HIV-2 provirus or strain thereof Alternatively, the transgene 
can be a modified form of a wild-type provirus, such as a provirus having 
a deletion, substitution or addition of at least one nucleotide to the 
viral genome. For example, a provirus can be modified by replacing the 
transcriptional control element in an LTR of the HIV provirus with another 
transcriptional control element, for example to alter the tropism of the 
virus. A provirus can also be modified by deleting a portion of, or 
mutating, at least one HIV gene, to thereby inactivate at least one HIV 
protein, e.g, gag, pol, env, or tat. In another embodiment, the HIV 
transgene is non-infectious. For example, an HIV provirus can be rendered 
non-infectious by deleting a portion of gag and pol or by mutating at 
least one LTR of the provirus. 
In a further aspect, the invention features non-human animal cells 
containing a lentivirus transgene, e.g., an HIV transgene. For example, 
the animal cell (e.g. somatic cell or germ cell (i.e. egg or sperm)) can 
be obtained from a lentivirus transgenic animal. Transgenic somatic cells 
or cell lines can be used, for example, in drug screening assays. 
Transgenic germ cells, on the other hand, can be used in generating 
transgenic progeny, as described above. 
In yet further aspects, the invention features methods for using the 
non-human transgenic animals, cells and cell lines of the invention for 
investigating molecular and cellular mechanisms of lentiviral mediated 
pathogenesis (e.g. the molecular and cellular mechanisms of the skin 
lesions, CNS disturbances, heart and kidney disease, which is associated 
with human HIV infection); as well as for identifying compounds and 
vaccines for treating and/or preventing lentivirus (e.g. HIV) infection 
and disease (e.g. AIDS) development. 
A preferred in vitro assay for identifying molecular antagonists which, for 
example, interfere with a lentivirus ligand-receptor interaction, as well 
as molecular agonist which, for example, function by activating a 
lentivirus protein (e.g. receptor) is comprised of the steps of: (a) 
incubating transgenic cells expressing a protein (e.g. receptor) known to 
be involved in lentivirus infection with a test compound; and (b) 
detecting the interaction between the lentivirus protein and the test 
compound, wherein the presence of an interaction indicates that the test 
compound may be an inhibitor of lentivirus infection. In another 
embodiment, in step (a), the test compound is incubated with the 
transgenic cell in the presence of a compound, which is a binding partner 
(e.g. a receptor ligand) to the expressed protein and the interaction 
between the test compound and the lentivirus protein or between the 
lentivirus binding partner and the lentivirus protein is detected. 
In other embodiments, cell based assays can be used to identify compounds 
which modulate expression of a lentivirus gene, modulate translation of a 
lentivirus mRNA, or which modulate the stability of a lentivirus mRNA or 
protein. A preferred assay comprises the steps of: (a) incubating a 
transgenic cell, which expresses a particular lentivirus protein with a 
test compound; and (b) comparing the amount of the lentivirus protein 
produced to that produced by the same cell which has not been contacted 
with the test compound. 
In a further embodiment, the effect of a test compound on transcription of 
a particular lentivirus gene can be determined by a transfection assay, 
which uses a reporter gene operatively linked to at least a portion of the 
promoter of a lentivirus gene. 
A preferred in vivo assay for identifying a compound which is useful for 
treating or preventing a disease or condition associated with lentivirus 
infection is comprised of the steps of. a) administering a test compound 
to a lentivirus transgenic animal; and (b) observing at least one 
phenotype associated with infection by the lentivirus, wherein a change in 
phenotype indicates that the test compound is capable of treating or 
preventing the disease or condition. In a preferred embodiment for 
identifying an effective vaccine, the transgenic non-human animal is made 
with an infectious lentivirus transgene, the compound is a lentivirus 
antigen or combination of antigens and the phenotype is an immune 
response. In a particularly preferred embodiment for identifying effective 
HIV vaccines, the transgenic non-human animal is made with an infectious 
HIV transgene, alone or in conjunction with a transgene encoding a CD4 
receptor (e.g. a human CD4 receptor) and/or an HIV co-receptor transgene 
(e.g. CCR or CXCR4). 
In a further aspect, the invention features methods for treating subjects 
infected with a lentivirus or preventing infection by a lentivirus, 
comprising administering to the subject an effective amount of a compound 
identified according to an assay of the invention. 
Other aspects of the invention are described below or will be apparent to 
those skilled in the art in light of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION 
General 
The invention is based at least in part on the generation of HIV transgenic 
rats, which develop characteristic AIDS symptoms and that express the HIV 
envelope protein, gp120. 
Definitions 
For convenience, the meaning of certain terms and phrases employed in the 
specification, examples and appended claims are provided below. 
"Antigen" refers to a protein, polypeptide, peptide or other molecule, 
which is capable of eliciting an immune response when administered to a 
vertebrate. 
"Animal line" refers to a group of animals that are direct descendants of 
one founder animal and which bear one or more transgenes stably integrated 
into one or more loci in their germline. 
A "DNA construct" refers to a DNA molecule comprising a transgene. 
"Founder" generally refers to a first transgenic animal, which has been 
obtained from any of a variety of methods, e.g., pronuclei injection. 
"Genome" is intended to include the entire DNA complement of an organism, 
including the nuclear DNA component, chromosomal or extrachromosomal DNA, 
as well as the cytoplasmic domain (e.g., mitochondrial DNA). The genome of 
a eukaryotic organisms, in contrast to bacterial and viral organisms, is 
usually arranged into chromosomes within the cell nucleus. 
"gp-120 is expressed on the surface of a cell" or "a cell expressing gp-120 
on its surface" refers to a cell which contains at least one molecule of 
gp-120 on its surface, preferably at least about 10, at least about 100, 
at least about 1000, at least about 10,000 or at least about 100,000 
gp-120 molecules on its surface. Preferred cells expressing gp-120 are 
those on which gp-120 can be detected by flow cytometry using an 
anti-gp120 antibody. Other preferred cells expressing gp-120 are cells 
which have a biological activity typical of cells having gp-120 on their 
surface, e.g., interaction with CD4 and/or which are capable of syncytium 
formation. 
"Heterologous DNA", which is used interchangeably with "exogenous DNA" 
refers to DNA that is not naturally present in the cell. 
The term "HIV" is used interchangeably herein with the terms "LAV", 
"LAV-2", "HTLV-III", and "ARV" to refer to human immunodeficiency virus 
(HIV). HIV includes both type 1 and type 2 human immunodeficiency viruses 
and their strains, unless it is used within the context of a specific 
embodiment related to type 1 or type 2 virus. The terms "HIV-1" and 
"HIV-2" are used to distinguish the type 1 virus and its strains from the 
type 2 virus and its strains. The HIV-1 and HIV-2 genomes, and the DNA 
sequences of HIV-1 and HIV-2, and respective strains are further described 
herein, as well as in the publication "Human Retrovirus And AIDS 1991", 
Eds. G. Myer et al., Theoretical Biology and Biophysics, Los Alamos 
National Laboratory, Los Almos, N. Mex., 87545, USA. Nucleotide sequences 
of HIV strains can be found in Genbank under the following Accession Nos: 
1) HIV-1: K03455, M19921, K02013, M38431, M38429, K02007 and M17449; 2) 
HIV-2: M30502, J04542, M30895, J04498, M15390, M31113 and L07625 from J. 
M. Coffin, S. H. Hughes, and H. E. Varmus, "Retroviruses" Cold Spring 
Harbor Laboratory Press, 1997, p 804). In addition to HIV sequences 
available from Genbank as described above, HIV sequences can also be 
found, e.g., in the HIV sequence database publically available at 
hiv-web.lanl.gov. A map of the HIV-1 genome and transcripts can be found, 
e.g., in J. M. Coffin, S. H. Hughes, and H. E. Varmus, "Retroviruses" Cold 
Spring Harbor Laboratory Press, 1997, p803). Set forth in Table 1 is the 
name and nucleotide location of the major genes of HIV-1 (from Coffin et 
al., supra pp 802, 804): 
TABLE 1 
______________________________________ 
name nucleotides 
comments 
______________________________________ 
R 1-96 Repeat is a short sequence containing the 
transactivator response region (TAR, i.e., Tat 
Responsive) 
U5 97-181 
PBS 182-199 
gag 336-1836 encodes Pr55 Gag 
pro 1637-2099 encodes a Pr160 Gag-Pro-Pol precursor 
pol 2102-4640 pol gene products are synthesized as part of Pr160 
vif 4587-5163 encodes p23 Vif protein 
vpr 5105-5339 encodes p15 Vpr protein 
tat 5377-5591 encodes p14 Tat protein; binds to the Tat 
7925-7968 region of R 
rev 5516-5591 encodes p19 Rev protein 
7925-8197 
vpu 5608-5854 encodes p16 Vpu protein 
env 5771-8339 encodes the gPr160 Env precursor 
nef 8343-8710 encodes p27 Nef 
PPT 8615-8630 serves as principal primer for plus strand synthesis 
U3 8631-9085 
R 9086-9181 
______________________________________ 
Amino acid sequences of HIV-1 proteins can also be found in Genbank under 
the following Accession Nos.(from Coffin et al., supra p 804): gag-P04591; 
pol-P04585; env-p04578; vif-p03401; vpr-p05926; vpu-p05919; tat-p04608; 
rev-p04618; and nef-p0460 1. HIV-2 has basically the same molecular 
organization as HIV-1. However, contrary to HIV-1, HIV-2 contains a gene 
called vpx, which encodes a 14 kDa protein of unknown function. Another 
difference is that HIV-2 does not contain the vpu gene, which is present 
in the HIV-1 genome. Another difference between HIV-1 and HIV-2 is the 
presence of a large insertion in the HIV-2 rev gene. There are also 
significant differences between HIV-1 and HIV-2 rev in addition to this 
insertion. 
In addition, a human T cell line that produces HIV is available under ATCC 
Designation No. CRL-8543. A vector containing the full length HIV-1 genome 
is available under ATCC Designation No. 53069. DNA encoding specific HIV 
genes is also available from the ATCC, e.g., a clone of human TAR (HIV) 
RNA binding protein 1 is available under ATCC Designation No. 107237 and a 
DNA encoding env-3 from HIV-1 is available under ATCC Designation No. 
53072. 
"Homology" or "identity" or "similarity" refers to sequence similarity 
between two peptides or between two nucleic acid molecules. Homology can 
be determined by comparing a position in each sequence which may be 
aligned for purposes of comparison. When a position in the compared 
sequence is occupied by the same base or amino acid, then the molecules 
are identical at that position. A degree of homology or similarity or 
identity between nucleic acid sequences is a function of the number of 
identical or matching nucleotides at positions shared by the nucleic acid 
sequences. Two DNA sequences are "substantially homologous" or 
"substantially similar" when at least about 75% (preferably at least about 
80%, and most preferably at least about 90 or 95%) of the nucleotides 
match over the defined length of the DNA sequences. Sequences that are 
substantially homologous can be identified by comparing the sequences 
using standard software available in sequence data banks, or in a Southern 
hybridization experiment under, for example, stringent conditions as 
defined for that particular system. Defining appropriate hybridization 
conditions is within the skill of the art. See, e.g., Maniatis et al., 
supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, 
supra. A degree of identity of amino acid sequences is a function of the 
number of identical amino acids at positions shared by the amino acid 
sequences. A degree of homology or similarity of amino acid sequences is a 
function of the number of amino acids, i.e. structurally related, at 
positions shared by the amino acid sequences. An "unrelated" or 
"non-homologous" sequence shares less than 40% identity, though preferably 
less than 25% identity, with one of the sequences of the present 
invention. Two amino acid sequences are "substantially homologous" or 
"substantially similar" when greater than 70% of the amino acids are 
identical, or functionally identical. Preferably, the similar or 
homologous sequences are identified by alignment using, for example, the 
GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 
7, Madison, Wis.) pileup program. 
An "infectious" virus or virus particle refers to a virus that is capable 
of replicating and producing new viral particles when it infects an 
appropriate cell. 
An "inbred animal line" is intended to refer to animals which are 
genetically identical at all endogenous loci. 
The term "isolated" as used herein with respect to nucleic acids, such as 
DNA or RNA, refers to molecules separated from other DNAs, or RNAs, 
respectively, that are present in the natural source of the macromolecule. 
The term isolated as used herein also refers to a nucleic acid or peptide 
that is substantially free of cellular material, viral material, or 
culture medium when produced by recombinant DNA techniques, or chemical 
precursors or other chemicals when chemically synthesized. Moreover, an 
"isolated nucleic acid" is meant to include nucleic acid fragments which 
are not naturally occurring as fragments and would not be found in the 
natural state. The term "isolated" is also used herein to refer to 
polypeptides which are isolated from other cellular proteins and is meant 
to encompass both purified and recombinant polypeptides. 
"Lentiviruses" include primate lentiviruses, e.g., human immunodeficiency 
virus types 1 and 2 (HIV-1/HIV-2); simian immunodeficiency virus (SIV) 
from Chimpanzee (SIVcpz), Sooty mangabey (SIVsmm), African Green Monkey 
(SIVagm), Syke's monkey (SIVsyk), Mandrill (SIVmnd) and Macaque (SIVmac). 
Lentiviruses also include feline lentiviruses, e.g., Feline 
immunodeficiency virus (FIV); Bovine lentiviruses, e.g., Bovine 
immunodeficiency virus (BIV); Ovine lentiviruses, e.g., Maedi/Visna virus 
(MVV) and Caprine arthritis encephalitis virus (CAEV); and Equine 
lentiviruses, e.g., Equine infectious anemia virus (EIAV). All 
lentiviruses express at least two additional regulatory proteins (Tat, 
Rev) in addition to Gag, Pol, and Env proteins. Primate lentiviruses 
produce other accessory proteins including Nef, Vpr, Vpu, Vpx, and Vif. 
Generally, lentiviruses are the causative agents of a variety of disease, 
including, in addition to immunodeficiency, neurological degeneration, and 
arthritis. Nucleotide sequences of the various lentiviruses can be found 
in Genbank under the following Accession Nos. (from J. M. Coffin, S. H. 
Hughes, and H. E. Varmus, "Retroviruses" Cold Spring Harbor Laboratory 
Press, 199,7 p 804): 1) HIV-1: K03455, M19921, K02013, M3843 1, M38429, 
K02007 and M17449; 2) HIV-2: M30502, J04542, M30895, J04498, M15390, 
M31113 and L07625; 3) SIV:M29975, M30931, M58410, M66437, L06042, M33262, 
M19499, M32741, M31345 and L03295; 4) FIV: M25381, M36968 and Ul 1820; 
5)BIV. M32690; 6)E1AV: M16575, M87581 and U01866; 6)Visna: M10608, M51543, 
L06906, M60609 and M60610; 7) CAEV: M33677; and 8) Ovine lentivirus M31646 
and M34193. Lentiviral DNA can also be obtained from the American Type 
Culture Collection (ATCC). For example, feline immunodeficiency virus is 
available under ATCC Designation No. VR-2333 and VR-3112. Equine 
infectious anemia virus A is available under ATCC Designation No. VR-778. 
Caprine arthritis-encephalitis virus is available under ATCC Designation 
No. VR-905. Visna virus is available under ATCC Designation No. VR-779. 
"Nucleic acid" refers to polynucleotides such as deoxyribonucleic acid 
(DNA), and, where appropriate, ribonucleic acid (RNA). The term should 
also be understood to include, as equivalents, analogs of either RNA or 
DNA made from nucleotide analogs, and, as applicable to the embodiment 
being described, single (sense or antisense) and double-stranded 
polynucleotides. 
"Non-infectious" virus or virus particle refers to a virus that is 
incapable of producing new viral particles even when it infects an 
appropriate cell. A non-infectious human immunodeficiency virus typically 
has a deletion in gag and/or pol and is thereby incapable of replicating 
and encapsidating the viral DNA. 
"Phenotype" refers to an observable property of an organism (in contrast to 
the genotype, i.e. genetic composition of the organism). 
The terms "protein", "polypeptide" and "peptide" are used interchangeably 
herein when referring to a gene product. 
The term "proviral" DNA refers to a form of a virus that is integrated into 
the genetic material of a host cell and by replicating with it can be 
transmitted from one cell generation to the next. 
"Small molecule" as used herein, is meant to refer to a composition, which 
has a molecular weight of less than about 5 kD and most preferably less 
than about 4 kD. Small molecules can be nucleic acids, peptides, 
polypeptides, peptidomimetics, carbohydrates, lipids or other organic 
(carbon containing) or inorganic molecules. Many pharmaceutical companies 
have extensive libraries of chemical and/or biological mixtures, often 
fungal, bacterial, or algal extracts, which can be screened with any of 
the assays of the invention to identify compounds that inhibit viral gene 
expression, virus replication, and/or viral production. 
DNA "regulatory elements" include transcriptional and translational control 
elements, such as promoters, enhancers, silencers, terminators, and the 
like, that provide for translation or expression of a nucleic acid. In 
eukaryotic cells, polyadenylation signals are control sequences. 
The phrase "therapeutically effective amount" as used herein refers to an 
amount sufficient to improve by at least about 15 percent, preferably by 
at least about 50 percent, more preferably by at least about 90 percent, 
and most preferably by about 100% (i.e., cure) a medical condition or 
symptoms thereof in a subject. Alternatively the therapeutically effective 
amount can be an amount sufficient to reduce by at least about 15 percent, 
preferably by at least about 50 percent, more preferably by at least about 
90 percent, and most preferably by about 100% the viral load, expression 
of a gene, e.g, a viral gene, or viral replication. 
A cell has been "transfected" by exogenous or heterologous DNA when such 
DNA has been introduced into the cell. A cell has been "transformed" by 
exogenous or heterologous DNA when the transfected DNA effects a 
phenotypic change. Preferably, the transforming DNA should be integrated 
(covalently linked) into chromosomal DNA making up the genome of the cell. 
The term "transgene" broadly refers to any nucleic acid that is introduced 
into an animal's genome, including but not limited to genes or DNA having 
sequences which are perhaps not normally present in the genome, genes 
which are present, but not normally transcribed and translated 
("expressed") in a given genome, or any other gene or DNA which one 
desires to introduce into the genome. This may include genes which may 
normally be present in the nontransgenic genome but which one desires to 
have altered in expression, or which one desires to introduce in an 
altered or variant form. A transgene can include one or more 
transcriptional regulatory sequences and any other nucleic acid, such as 
introns, that may be necessary for optimal expression of a selected 
nucleic acid. A preferred transgene of the invention is a viral transgene, 
e.g., a lentiviral transgene. A transgene can be as few as a couple of 
nucleotides long, but is preferably at least about 50, 100, 150, 200, 250, 
300, 350, 400, or 500 nucleotides long or even longer and can be, e.g., an 
entire viral genome. A transgene can be coding or non-coding sequences, or 
a combination thereof A transgene usually comprises a regulatory element 
that is capable of driving the expression of one or more transgenes under 
appropriate conditions. A "lentiviral transgene" refers to a nucleic acid 
comprising a nucleotide sequence encoding at least one lentiviral protein 
or biologically active portion thereof. An "HIV transgene" refers to a 
nucleic acid comprising a nucleotide sequence encoding at least one HIV 
protein or biologically active portion thereof 
A "transgenic animal" refers to any animal, preferably a non-human mammal 
(e.g. mouse, rat, rabbit, squirrel, hamster, rabbits, guinea pigs, pigs, 
micro-pigs, prairie, baboons, squirrel monkeys and chimpanzees, etc), bird 
or an amphibian, in which one or more cells contain heterologous nucleic 
acid introduced by way of human intervention, such as by transgenic 
techniques well known in the art. The nucleic acid is introduced into the 
cell, directly or indirectly, by introduction into a precursor of the 
cell, by way of deliberate genetic manipulation, such as by microinjection 
or by infection with a recombinant virus. The term genetic manipulation 
does not include classical cross-breeding, or in vitro fertilization, but 
rather is directed to the introduction of a recombinant DNA molecule. This 
molecule may be integrated within a chromosome, or it may be 
extrachromosomally replicating DNA. In the typical transgenic animals 
described herein, the transgene causes cells to express a viral gene. 
However, transgenic animals in which the transgene is silent are also 
contemplated, as for example, the FLP or CRE recombinase dependent 
constructs. 
The term "treating" as used herein is intended to encompass curing as well 
as ameliorating at least one symptom of the condition or disease. 
A "vaccine" refers to a preparation containing at least one lentiviral 
antigen, which can be administered to a subject to produce or artificially 
increase immunity to a disease, which is caused by or contributed to by a 
lentivirus. In addition to the at least one antigen, the vaccine can 
optionally comprise a pharmaceutically acceptable carrier and/or an 
adjuvant. 
The term "wild-type viral gene or genome" refers to a viral gene or genome 
as it is found in nature, i.e., which has not been manipulated by man. 
Thus, there may exist several wild-type genomes for each type of virus. 
Lentiviral Transgenes and Trans genic Animals Produced Therefrom 
The invention provides for transgenic non-human animals comprising a 
lentivirus transgene, (e.g., an HIV transgene). The lentiviral construct 
can be an infectious virus, which is capable of replicating and producing 
viral particles. An example of an infectious HIV-1 DNA includes the DNA 
construct referred to as pNL4-3 (Adachi et al. (1986) J. Virol. 59:284 and 
Leonard et al. (1988) Science 242:1665; Genbank Accession No. M19921). 
Another infectious HIV-1 proviral construct is pNL4-32 (Strebel et al. 
(1987) J. Virol. 328:728 and Leonard et al., supra). Transgenic non-human 
animals made with infectious HIV transgenes, alone or in conjunction with 
a transgene encoding a CD4 receptor (e.g. the human CD4 receptor) and/or 
an HIV co-receptor transgene (e.g. CCR5 or CXCR4) can produce infectious 
viral particles, which infect host cells, and therefore are particularly 
preferred for developing effective HIV vaccines and therapeutics. 
Non-human transgenic animals, which are noninfectious and therefore 
potentially safer for use, can be generated using transgenes comprised of 
non-infectious viral DNA, i.e., viral DNA which does not result in the 
formation of viral particles upon infection of a host cell. For example, a 
non-infectious viral DNA can have a deletion or other type of mutation in 
any coding region or regulatory region sufficient to impair viral nucleic 
acid replication, and/or assembly of virions. The deletion can inhibit 
production of, or inactivate, one or more of the proteins selected from 
the group consisting of a nucleocapsid-core factor (e.g., gag), reverse 
transcriptase, protease, integrase, ribonuclease, and transcriptional 
activator (e.g., tat). An example of an HIV-1 provirus that is 
non-infectious is the pNL4-3 :dI443 vector, which is derived from the 
infectious pNL4-3 vector by deletion of a 3.1 kb sequence overlapping gag 
and pol (sequences between the SphI and BalI sites at bases 1443-4556), 
but containing env and the other accessory genes tat, nef, vif, vpr, and 
vpu, together with the 5' and 3' long terminal repeats (LTRs). As 
described in the following Examples, pNL4-3:d1443 has been used to produce 
transgenic rats, which model human AIDS. 
Other non-infectious HIV DNA can be obtained by deleting portions of one or 
both LTRs. For example, the HIV DNA sequence can be prepared by digesting 
a plasmid clone containing the DNA sequence of HIV-1 with a restriction 
enzyme that cleaves the HIV proviral DNA sequence at sites proximal to its 
5' and 3' ends, thereby removing essential controlling sequences, to yield 
a proviral DNA sequence truncated at both ends, so that the eventual RNA 
expression from the cleaved fragment is rendered non-infectious, but still 
includes those elements required for the eventual production of at least 
some viral proteins. In other words, the HIV genome is modified to lack 
the sequences necessary for reverse transcription, integration and/or 
transcription. The extent to which the 5' and 3' ends must be truncated to 
render the RNA non-infectious can be determined by standard methods (e.g. 
by transforming the fragment so obtained into a genomic equivalent of 
HIV-1 and testing the resulting virus for cytopathic activity). As an 
example, the SacI restriction enzyme can be used to cleave the pBH10 
plasmid [B. H. Hahn et al., Nature, 312, 166 (1984)] to yield an HIV-1 
genome deleted of the 5' LTR and/or a portion of its 3' LTR. 
Non-infectious HIV proviral DNA deleted 5' and/or 3' are further described 
in U.S. Pat. No. 5,574,206 by Jolicoeur. 
Another method for obtaining a non-infectious HIV proviral DNA sequence 
involves truncating the HIV genomic DNA fragment from its 5' end to a 
point on the untranslated 5' leader sequence located between about 50 
nucleotides downstream from the 5' LTR, but not including the nucleotide 
marking the beginning of the splice donor sequence; and truncating the 
same HIV DNA fragment from its 3' end sequence to a point located 
downstream of the nef gene, so that the complete encoding sequence of the 
nef gene is retained and sequences required for virus replication (i.e. 
the U5, R and part of the U3 sequences) are deleted. 
Transgenic animals exhibiting tissue specific expression can be generated, 
for example, by inserting a tissue specific regulatory element, such as an 
enhancer, into the viral transgene. For example, one of the LTRs or a 
portion thereof can be replaced with another promoter and/or enhancer, 
e.g., a CMV or a Moloney murine leukemia virus (MLV) promoter and/or 
enhancer. For example, the proviral HIV DNA is pNL4-3, in which the two 
NF-.kappa.B binding motifs of the HIV core enhancer sequences from the 
Moloney murine Leukemia Virus (Mo-MuLV) LTR by M13 mutagenesis (deleting 
nucleotides -129 to -74, with respect to the HIV mRNA cap site and 
replacing them with nucleotides -365 to -40 from the Mo-MuLV). This 
construct is further described (as pHm4-3) in Dickie et al. (1996) AIDS 
Res. Human Retroviruses 12:177, which also describes transgenic mice 
containing this construct. 
An LTR of a proviral genome, e.g, HIV proviral genome, can also be replaced 
with a mouse mammary tumor virus (MMTV) LTR, which is known to be tissue 
specific toward various epithelial and hematopoietic tissues, some of 
which naturally support lentivirus (and especially HIV) replication. (for 
an example of such a construct, see, e.g., U.S. Pat. No. 5,574,206 issued 
Nov. 12, 1996 to Jolicoeur). 
Alternatively, non-human transgenic animals that only express HIV 
transgenes in the brain can be generated using brain specific promoters 
(e.g. myelin basic protein (MBP) promoter, the neurofilament protein 
(NF-L) promoter, the gonadotropin-releasing hormone promoter, the 
vasopressin promoter and the neuron-specific enolase promoter, see So 
Forss-Petter et al., Neuron, 5, 187, (1990). Such animals can provide a 
useful in vivo model to evaluate the ability of a potential anti-HIV drug 
to cross the blood-brain barrier. Other target cells for which specific 
promoters can be used are, for example, macrophages, T cells and B cells. 
Other tissue specific promoters are well-known in the art, see e.g. 
R.Jaenisch, Science, 240, 1468 (1988). 
Non-human transgenic animals containing an inducible lentiviral transgene 
(infectious or noninfectious) can be generated using inducible regulatory 
elements (e.g. metallothionein promoter), which are well-known in the art. 
Lentiviral gene expression can then be initiated in these animals by 
administering to the animal a compound which induces gene expression (e.g. 
heavy metals). Another preferred inducible system comprises a 
tetracycline-inducible transcriptional activator (U.S. Pat. No. 5,654,168 
issued Aug. 5, 1997 to Bujard and Gossen and U.S. Pat. No. 5,650,298 
issued Jul. 22, 1997 to Bujard et al.). 
Double, triple or multimeric transgenic animals, comprise at least one 
other transgene in addition to the lentiviral transgene. In a preferred 
embodiment, the animal comprises an HIV transgene and a transgene encoding 
human CD4 protein or a portion thereof sufficient for HIV infection of 
target cells. The nucleotide sequence of cDNA encoding human CD4 can be 
found, e.g., in Maddon et al. (1985) Cell 42:93. Mice containing a CD4 
transgene are described, e.g., in Wang et al., (1994) Eur. J. Immunol., 
24: 1553. A human CD4 transgenic rabbit has been described, e.g, in PCT 
application No. PCT/FR93/00598 (WO 94/00568) by Mehtali et al. 
In another preferred embodiment, the animal comprises an HIV transgene and 
a transgene, which expresses a coreceptor (e.g. CCR5 or CXCR4). In a 
further preferred embodiment, the animal comprises an HIV transgene and a 
transgene (e.g. mutant gene), which is involved in a disease or condition 
that is associated with AIDS (e.g. hypertension, Kaposi's sarcoma, 
cachexia, etc.). For example, the transgenic animals of the invention can 
be crossed with hypertensive rats of the transgenic rat strain 
TGR(mREN2)27 harboring the murine Ren-2 gene or transgenic rats comprising 
a transgene encoding human angiotensinogen and/or human renin (U.S. Pat. 
No. 5,731,489). These transgenic rats develop fulminant hypertension at an 
early age despite low levels of renin in plasma and kidney (described in, 
e.g., Lee et al. (1996) Am J Physiol 270: E919). 
Where one or more genes encoding a protein are used as transgenes, it may 
be desirable to operably link the gene to an appropriate regulatory 
element, which will allow expression of the transgene. Regulatory 
elements, e.g., promoters, enhancers, (e.g. inducible or constitutive) or 
polyadenylation signals are well known in the art. Regulatory sequences 
can be endogenous regulatory sequences, i.e., regulatory sequences from 
the same animal species as that in which it is introduced as a transgene. 
The regulatory sequences can also be the natural regulatory sequence of 
the gene that is used as a transgene. Accordingly, regulatory elements for 
a CD4 transgene can be the natural CD4 regulatory elements and can include 
5' flanking sequence of the CD4 gene comprising promoter and enhancer 
sequences. 
Alternatively, a transgene can be placed under the control of an exogenous 
regulatory element, i.e. a regulatory element, which is not the normal 
regulatory element of the transgene. For example, a human CD4 transgene 
can be placed under the control of a promoter that is functional in 
specific cell types, e.g., in T lymphocytes. An exemplary promoter for use 
in transgenic rats is the rat CD2 promoter. Alternatively, the 
transcriptional regulatory element can be a viral promoter, e.g, the MMTV 
LTR. 
The transgene also preferably contains a polyadenylation signal (poly A 
addition sequence), which can comprise one or a tandem of two to four of 
the known poly A addition signal sequences, such as those derived from the 
SV40 genome, the casein 3' untranslated region or other 3' untranslated 
sequences known in the art. A convenient and readily available source for 
the poly A addition signal is the commercially available pSV2neo vector 
from which the SV40 poly A addition signal sequence can be cleaved. The 
transgene can be prepared using techniques known in the art; for example 
see J. Sambrook et al., "Molecular Cloning: A Laboratory Manual", 2nd ed, 
Vols 1 to 3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 
N.Y., USA, 1989. 
Constructs for use as transgenes can first be tested for expression in cell 
lines. Where the transcriptional control elements in the construct are 
those from a virus, e.g., HIV, it may be desirable to use a test cell line 
of the same type as that which is naturally infected by the virus. For 
example, when testing a construct derived from an HIV provirus, it may be 
desirable to use a cell line in which HIV is expressed and is preferably 
capable of replicating, e.g., T cell lines. Examples of cell lines in 
which HIV is known to replicate include primary human PBMC, isolated 
macrophages, isolated CD4+ T cells and cultured human cell lines, such as 
HeLa and H9. Expression of the transgene and/or production of viral 
particles can be detected as further set forth herein. 
Production of transgenic non-human animals 
In general, transgenic animal lines can be obtained by generating 
transgenic animals having incorporated into their genome at least one 
transgene, selecting at least one founder from these animals and breeding 
the founder or founders to establish at least one line of transgenic 
animals having the selected transgene incorporated into their genome. 
Animals for obtaining eggs or other nucleated cells (e.g. embryonic stem 
cells) for generating transgenic animals can be obtained from standard 
commercial sources such as Charles River Laboratories (Wilmington, Mass.), 
Taconic (Germantown, N.Y.), Harlan Sprague Dawley (Indianapolis, Ind.). 
Eggs can be obtained from suitable animals, e.g., by flushing from the 
oviduct or using techniques described in U.S. Pat. No. 5,489,742 issued 
Feb. 6, 1996 to Hammer and Taurog; U.S. Pat. No. 5,625,125 issued on Apr. 
29, 1997 to Bennett et al.; Gordon et al., 1980, Proc. Natl. Acad. Sci. 
USA 77:7380-7384; Gordon & Ruddle, 1981, Science 214: 1244-1246; U.S. Pat. 
No. 4,873,191 to T. E. Wagner and P. C. Hoppe; U.S. Pat. No. 5,604,131; 
Armstrong, et al. (1988) J. of Reproduction, 39:511 or PCT application No. 
PCT/FR93/00598 (WO 94/00568) by Mehtali et al. Preferably, the female is 
subjected to hormonal conditions effective to promote superovulation prior 
to obtaining the eggs. 
Many techniques can be used to introduce DNA into an egg or other nucleated 
cell, including in vitro fertilization using sperm as a carrier of 
exogenous DNA ("sperm-mediated gene transfer", e.g., Lavitrano et al., 
1989, Cell 57: 717-723), microinjection, gene targeting (Thompson et al., 
1989, Cell 56: 313-321), electroporation (Lo, 1983, Mol. Cell. Biol. 3: 
1803-1814), transfection, or retrovirus mediated gene transfer (Van der 
Putten et al., 1985, Proc. Natl. Acad. Sci. USA 82: 6148-6152). For a 
review of such techniques, see Gordon (1989), Transgenic Animals, Intl. 
Rev. Cytol. 115:171-229. 
Except for sperm-mediated gene transfer, eggs should be fertilized in 
conjunction with (before, during or after) other transgene transfer 
techniques. A preferred method for fertilizing eggs is by breeding the 
female with a fertile male. However, eggs can also be fertilized by in 
vitro fertilization techniques. 
Fertilized, transgene containing eggs can than be transferred to 
pseudopregnant animals, also termed "foster mother animals", using 
suitable techniques. Pseudopregnant animals can be obtained, for example, 
by placing 40-80 day old female animals, which are more than 8 weeks of 
age, in cages with infertile males, e.g., vasectomized males. The next 
morning females are checked for vaginal plugs. Females who have mated with 
vasectomized males are held aside until the time of transfer. 
Recipient females can be synchronized, e.g. using GNRH agonist (GnRH-a): 
des-gly10, (D-Ala6)-LH-RH Ethylamide, SigmaChemical Co.,St. Louis, Mo. 
Alternatively, a unilateral pregnancy can be achieved by a brief surgical 
procedure involving the "peeling" away of the bursa membrane on the left 
uterine horn. Injected embryos can then be transferred to the left uterine 
horn via the infundibulum. Potential transgenic founders can typically be 
identified immediately at birth from the endogenous litter mates. For 
generating transgenic animals from embryonic stem cells, see e.g. 
Teratocarcinomas and embryonic stem cells, a practical approach, ed. E. J. 
Robertson, (IRL Press 1987) or in Potter et al Proc. Natl. Acad. Sci. USA 
81, 7161 (1984), the teachings of which are incorporated herein by 
reference. 
Founders that express the gene can then bred to establish a transgenic 
line. Accordingly, founder animals can be bred, inbred, crossbred or 
outbred to produce colonies of animals of the present invention. Animals 
comprising multiple transgenes can be generated by crossing different 
founder animals (e.g. an HIV transgenic animal and a transgenic animal, 
which expresses human CD4), as well as by introducing multiple transgenes 
into an egg or embryonic cell as described above. Furthermore, embryos 
from A-transgenic animals can be stored as frozen embryos, which are 
thawed and implanted into pseudo-pregnant animals when needed (See e.g. 
Hirabayashi et al. (1997) Exp Anim 46: 111 and Anzai (1994) Jikken Dobutsu 
43: 247). 
The present invention provides for transgenic animals that carry the 
transgene in all their cells, as well as animals that carry the transgene 
in some, but not all cells, i.e., mosaic animals. The transgene can be 
integrated as a single transgene or in tandem, e.g., head to head tandems, 
or head to tail or tail to tail or as multiple copies. 
The successful expression of the transgene can be detected by any of 
several means well known to those skilled in the art. Non-limiting 
examples include Northern blot, in situ hybridization of mRNA analysis, 
Western blot analysis, immunohistochemistry, and FACS analysis of protein 
expression. 
In particular, the expression of the gag protein (p55), the gag protein 
cleavage products p24 and p17, the envelope glycoprotein (gp160) and the 
envelope protein cleavage product gp120 can be detected using specific 
probes and/or antibodies. At least some of these antibodies are 
commercially available. For example, monospecific anti-gp120 reagent can 
be obtained, e.g., from Biochorm, Seromed Ref. D7324. Sheep anti-gp120 
serum of HIV-1 can also be obtained from the AIDS Research and Reference 
Program (catalog no. 192), National Institutes of Health, Bethesda, Md. 
Human monoclonal anti-gp120 antibodies are also described in U.S. Pat. No. 
5,695,927 issued Dec. 7, 1997 to Masuho et al. 
Animal tissue can also be analyzed directly, e.g., by preparing tissue 
sections. In some embodiments, it is preferable to fix the tissue, e.g., 
with paraformaldehyde or formalin. Tissue sections can be prepared frozen, 
or can be paraffin embedded. Slides of animal tissue can be used for 
immunohistochemistry, in vitro hybridization or for regular histology, 
e.g., hematoxylin and eosin staining. 
Virus can be quantitated by reverse transcriptase (RT) activity, as is 
well-known in the art. A change in viral load can also be determined by 
quantitative assays for plasma HIV RNA using quantitative RT-PCR as 
described, e.g, in Van Gemen et al. (1994) J. Viro. Methods 49:157; Chen 
et al. (1992) AIDS 6:533. Alternatively, viral load can be determined by 
assays for viral production from isolated PBMCs. Viral production from 
PBMCs is determined by cocultruring PBMCs from the subject with H9 cells 
and subsequent measurement of HIV titers using an ELISA assay for p24 
antigen levels (Popovic et al. (1984) Science 204:497; PCT/US97/11202 
(W097/49373) by Gallo et al.). To identify lymphoid cell types expressing 
viral RNA, peritoneal inflammatory macrophages derived from the transgenic 
animals can be cultured ex vivo and examined by Northern blot analysis. 
Disease Models and Drug Screening Assays 
The invention further provides methods for identifying (screening) or for 
determining the safety and/or efficacy of lentivirus therapeutics, i.e. 
compounds which are useful for treating and/or preventing the development 
of diseases or conditions, which are caused by, or contributed to by 
lentiviral infection (e.g. AIDS). In addition the assays are useful for 
further improving known anti-viral compounds, e.g, by modifying their 
structure to increase their stability and/or activity and/or toxicity. 
In vitro cellular assays 
Cells from the transgenic animals of the invention can be established in 
culture and immortalized to establish cell lines. For example, 
immortalized cell lines can be established from the livers of transgenic 
rats, as described in Bulera et al. (1997) Hepatology 25: 1192. Cell lines 
from other types of cells can be established according to methods known in 
the art." 
In one cell-based assay, cells expressing a lentivirus protein (e.g. 
receptor) on the outer surface of its cellular membrane can be incubated 
in the presence of a test compound alone or in the presence of a test 
compound and a lentivirus protein binding partner (e.g. a receptor ligand) 
and the interaction between the test compound and the lentivirus protein 
or between the lentivirus binding partner (preferably tagged) and the 
lentivirus protein can be detected, e.g., using a microphysiometer 
(McConnell et al. (1992) Science 257:1906). An interaction between the 
lentivirus protein and either the test compound or the lentivirus protein 
binding partner can be detected, (e.g. with a microphysiometer as a change 
in the acidification of the medium). This assay system thus provides a 
means of identifying molecular antagonists which, for example, function by 
interfering with a lentivirus ligand-receptor interaction, as well as 
molecular agonist which, for example, function by activating a lentivirus 
protein (e.g. receptor). 
Cell based assays can also be used to identify compounds which modulate 
expression of a lentivirus gene, modulate translation of a lentivirus 
mRNA, or which modulate the stability of a lentivirus mRNA or protein. 
Accordingly, a cell which is capable of expressing a particular lentivirus 
protein can be incubated with a test compound and the amount of the 
lentivirus protein produced in the cell medium can be measured and 
compared to that produced from a cell which has not been contacted with 
the test compound. The specificity of the compound for regulating the 
expression of the particular lentivirus gene can be confirmed by various 
control analyses, e.g., measuring the expression of one or more control 
genes. This type of cellular assay can be particularly useful for 
determining the efficacy of antisense molecules or ribozymes. 
In another embodiment, the effect of a test compound on transcription of a 
particular lentivirus gene can be determined by transfection experiments 
using a reporter gene, which is operatively linked to at least a portion 
of the promoter of a lentivirus gene. A promoter region of a gene can be 
isolated, e.g., from a genomic library according to methods known in the 
art. The reporter gene can be any gene encoding a protein which is readily 
quantifiable, e.g, the luciferase or CAT gene. Such reporter gene are well 
known in the art. 
In vivo assays in transgenic animals 
In addition to providing cells for in vitro assays, the transgenic animals 
themselves can be used in in vivo assays to identify lentiviral 
therapeutics. For example, the animals can be used in assays to identify 
compounds which reduce or inhibit any phase of the lentiviral life cycle, 
e.g., expression of one or more viral genes, activity of one or more viral 
proteins, glycosylation of one or more viral proteins, processing of one 
or more viral proteins, viral replication, assembly of virions, and/or 
budding of infectious virions. 
In an exemplary embodiment, the assay comprises administering a test 
compound to a transgenic animal of the invention and comparing a 
phenotypic change in the animal relative to a transgenic animal which has 
not received the test compound. For example, where the animal is an HIV 
transgenic animal, the phenotypic change can be the amelioration in an 
AIDS related complex (ARC), cataracts, inflammatory lesions in the central 
nervous system (CNV), a mild kidney sclerotic lesion, or a skin lesion, 
such as psoratic dermatitis, hyperkerstotic lesions, Kaposi's sarcoma or 
cachexia. The effect of a compound on inhibition of Kaposi's sarcoma can 
be determined, as described, e.g., in PCT/US97/11202 (WO97/49373) by Gallo 
et al. These and other HIV related symptoms or phenotypes are further 
described in Leonard et al. (1988) Science 242:1665. 
In yet another embodiment, the phenotypic change is the number of CD4+ T 
cells or the ratio of CD4+ T cells versus CD8+ T cells. In HIV infected 
humans as well as in HIV transgenic mice, analysis of lymph nodes indicate 
that the number of CD4+ T cells decreases and the number of CD8+ T cells 
increases. Numbers of CD4+ and CD8+ T cells can be determined, for 
example, by indirect immunofluorescence and flow cytometry, as described, 
e.g., in Santoro et al., supra. 
Alternatively, a phenotypic change, e.g. a change in the expression level 
of an HIV gene can be monitored. The HIV RNA can be selected from the 
group consisting of gag mRNA, gag-pro-pol mRNA, vif mRNA, vpr mRNA, tat 
mRNA, rev mRNA, vpu/env mRNA, nef mRNA, and vpx mRNA. The HIV protein can 
be selected from the group consisting of Pr55 Gag and fragments thereof 
(p17 MA, p24 CA, p7 NC, p1, p9, p6, and p2), Pr160 Gag-Pro-Pol, and 
fragments thereof (p10 PR, p51 RT, p66 RT, p32 IN), p23 Vif, p15 Vpr, p14 
Tat, p19 Rev, p16 Vpu, gPr 160 Env or fragments thereof (gp120 SU and 
gp41TM), p27 Nef, and p14 Vpx. The level of any of these mRNAs or proteins 
can be determined in cells from a tissue sample, such as a skin biopsy, as 
described in, e.g., PCT/US97/11202 (W097/49373) by Gallo et al. 
Quantitation of HIV mRNA and protein is further described elsewhere herein 
and also in, e.g., Dickie et al. (1996) AIDS Res. Human Retroviruses 
12:1103. In a preferred embodiment, the level of gp120 on the surface of 
PBMC is determined. This can be done, as described in the examples, e.g., 
by immunofluorescence on PBMC obtained from the animals. 
For example, the proteins expressed in the cells of a transgenic animal of 
the invention may include processed gag proteins resulting from the 
cleavage of the HIV-1 encoded gag-pol gene, the cleavage being effected by 
the HIV-1 encoded protease. Thus, in one embodiment, the invention 
provides a method for evaluating a test compound as a potential HIV-1 
protease inhibitor. In an exemplary embodiment, the method involves: (a) 
administering a test compound to the transgenic animal, and (b) examining 
the effect of the test compound on the expressed gag proteins in the 
animal by monitoring the expression levels of the proteins or RNAs. The 
presence of the RNA transcript and the presence or decrease (or 
inhibition) of the processed proteins in the cells serves as a means for 
evaluating HIV protease inhibitors. 
Likewise, since the presence of the gag and envelope proteins in the fluid 
and tissues of the transgenic animal denotes that the HIV regulatory 
protein, rev, is expressed, the present invention provides a method for 
evaluating a test compound as a potential inhibitor of rev function. In an 
exemplary embodiment, the method involves: (a) administering a test 
compound to the transgenic animal, and (b) examining the effect of the 
test compound on the expressed gag and envelope proteins and the gag 
protein cleavage products in the animal by monitoring the expression 
levels thereof 
A further phenotypic change is the production level or rate of viral 
particles in the serum and/or tissue of the animal. This can be 
determined, e.g., by determining reverse transcriptase (RT activity) or 
viral load as described elsewhere herein as well as in PCT/US97/11202 
(WO97/49373) by Gallo et al., such as by determining p24 antigen. 
Yet another phenotypic change, which can indicate HIV infection or AIDS 
progression is the production of inflammatory cytokines such as IL-6, IL-8 
and TNF-.alpha.; thus, efficacy of a compound as an anti-HIV therapeutic 
can be assessed by ELISA tests for the reduction of serum levels of any or 
all of these cytokines. 
A vaccine can be tested by administering a test antigen to a transgenic 
animal of the invention. The animal can optionally be boosted with the 
same or a different antigen. The production of viral particles or 
expression of viral proteins is then measured at various times following 
the administration of the test vaccine. A decrease in the amount of viral 
particles produced or viral expression will indicate that the test vaccine 
is efficient in reducing or inhibiting viral production and/or expression. 
The amount of antibody produced by the animal in response to the vaccine 
antigen can also be determined according to methods known in the art and 
provides a relative indication of the immunogenicity of the particular 
antigen. 
Therapeutic and Prophylactic Compounds 
Compounds identified above as being useful for preventing lentiviral 
infection and/or treating a lentiviral disease, can be, e.g. a nucleic 
acid (e.g DNA, RNA or PNA), protein, peptide, peptidomimetic, small 
molecule, or derivative thereof Preferred compounds are capable of binding 
to, and inhibiting transcription, translation or processing of a 
lentiviral RNA or protein. Examples include antisense, ribozyme or triplex 
nucleic acids, small molecule ligands, antibody or antibody-like binding 
fragments). Alternative compounds are competitive inhibitors of a protein 
involved in lentiviral infection, such as a portion of human CD4 
sufficient to bind to gp120 and interfere with binding of gp120 proteins 
on the surface of an infected cell or on the surface of a viral particle 
to a human CD4 molecule on the surface of cells. 
Toxicity and therapeutic efficacy of such compounds can be determined by 
standard pharmaceutical procedures in cell cultures or experimental 
animals, e.g., for determining the Ld5O (The Dose Lethal To 50% Of The 
Population) and the Ed.sub.50 (the dose therapeutically effective in 50% 
of the population). The dose ratio between toxic and therapeutic effects 
is the therapeutic index and it can be expressed as the ratio LD.sub.50 
/ED.sub.50. Compounds which exhibit large therapeutic induces are 
preferred. While compounds that exhibit toxic side effects may be used, 
care should be taken to design a delivery system that targets such 
compounds to the site of affected tissue in order to minimize potential 
damage to uninfected cells and, thereby, reduce side effects. 
The data obtained from the cell culture assays and animal studies can be 
used in formulating a range of dosage for use in humans. The dosage of 
such compounds lies preferably within a range of circulating 
concentrations that include the ED.sub.50 with little or no toxicity. The 
dosage may vary within this range depending upon the dosage form employed 
and the route of administration utilized. For any compound used in the 
method of the invention, the therapeutically effective dose can be 
estimated initially from cell culture assays. A dose may be formulated in 
animal models to achieve a circulating plasma concentration range that 
includes the IC.sub.50 (i.e., the concentration of the test compound which 
achieves a half-maximal inhibition of symptoms) as determined in cell 
culture. Such information can be used to more accurately determine useful 
doses in humans. Levels in plasma may be measured, for example, by high 
performance liquid chromatography. 
Pharmaceutical compositions for use in accordance with the present 
invention may be formulated in conventional manner using one or more 
physiologically acceptable carriers or excipients. Thus, the compounds and 
their physiologically acceptable salts and solvates may be formulated for 
administration by, for example, injection, inhalation or insufflation 
(either through the mouth or the nose) or oral, buccal, parenteral or 
rectal administration. 
For such therapy, the compounds of the invention can be formulated for a 
variety of loads of administration, including systemic and topical or 
localized administration. Techniques and formulations generally may be 
found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., 
Easton, Pa. For systemic administration, injection is preferred, including 
intramuscular, intravenous, intraperitoneal, and subcutaneous. For 
injection, the compounds of the invention can be formulated in liquid 
solutions, preferably in physiologically compatible buffers such as Hank's 
solution or Ringer's solution. In addition, the compounds may be 
formulated in solid form and redissolved or suspended immediately prior to 
use. Lyophilized forms are also included. 
For oral administration, the pharmaceutical compositions may take the form 
of, for example, tablets or capsules prepared by conventional means with 
pharmaceutically acceptable excipients such as binding agents (e.g., 
pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl 
methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or 
calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or 
silica); disintegrants (e.g., potato starch or sodium starch glycolate); 
or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated 
by methods well known in the art. Liquid preparations for oral 
administration may take the form of, for example, solutions, syrups or 
suspensions, or they may be presented as a dry product for constitution 
with water or other suitable vehicle before use. Such liquid preparations 
may be prepared by conventional means with pharmaceutically acceptable 
additives such as suspending agents (e.g., sorbitol syrup, cellulose 
derivatives or hydrogenated edible fats); emulsifying agents (e.g., 
lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, 
ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., 
methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may 
also contain buffer salts, flavoring, coloring and sweetening agents as 
appropriate. 
Preparations for oral administration may be suitably formulated to give 
controlled release of the active compound. For buccal administration the 
compositions may take the form of tablets or lozenges formulated in 
conventional manner. For administration by inhalation, the compounds for 
use according to the present invention are conveniently delivered in the 
form of an aerosol spray presentation from pressurized packs or a 
nebuliser, with the use of a suitable propellant, e.g., 
dichlorodifluoromethane, trichlorofluoromethane, 
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the 
case of a pressurized aerosol the dosage unit may be determined by 
providing a valve to deliver a metered amount. Capsules and cartridges of 
e.g., gelatin for use in an inhaler or insufflator may be formulated 
containing a powder mix of the compound and a suitable powder base such as 
lactose or starch. 
The compounds may be formulated for parenteral administration by injection, 
e.g., by bolus injection or continuous infusion. Formulations for 
injection may be presented in unit dosage form, e.g., in ampoules or in 
multi-dose containers, with an added preservative. The compositions may 
take such forms as suspensions, solutions or emulsions in oily or aqueous 
vehicles, and may contain formulatory agents such as suspending, 
stabilizing and/or dispersing agents. Alternatively, the active ingredient 
may be in powder form for constitution with a suitable vehicle, e.g., 
sterile pyrogen-free water, before use. 
The compounds may also be formulated in rectal compositions such as 
suppositories or retention enemas, e.g., containing conventional 
suppository bases such as cocoa butter or other glycerides. 
In addition to the formulations described previously, the compounds may 
also be formulated as a depot preparation. Such long acting formulations 
may be administered by implantation (for example subcutaneously or 
intramuscularly) or by intramuscular injection. Thus, for example, the 
compounds may be formulated with suitable polymeric or hydrophobic 
materials (for example as an emulsion in an acceptable oil) or ion 
exchange resins, or as sparingly soluble derivatives, for example, as a 
sparingly soluble salt. Other suitable delivery systems include 
microspheres which offer the possiblity of local noninvasive delivery of 
drugs over an extended period of time. This technology utilizes 
microspheres of precapillary size which can be injected via a coronary 
chatheter into any selected part of the e.g. heart or other organs without 
causing inflammation or ischemia. The administered therapeutic is slowly 
released from these microspheres and taken up by surrounding tissue cells 
(e.g. endothelial cells). 
Systemic administration can also be by transmucosal or transdermal means. 
For transmucosal or transdermal administration, penetrants appropriate to 
the barrier to be permeated are used in the formulation. Such penetrants 
are generally known in the art, and include, for example, for transmucosal 
administration bile salts and fusidic acid derivatives, in addition, 
detergents may be used to facilitate permeation. Transmucosal 
administration may be through nasal sprays or using suppositories. For 
topical administration, the oligomers of the invention are formulated into 
ointments, salves, gels, or creams as generally known in the art. A wash 
solution can be used locally to treat an injury or inflammation to 
accelerate healing. 
In situations in which the therapeutic is a gene, a gene delivery system 
can be introduced into a patient by any of a number of methods, each of 
which is familiar in the art. For instance, a pharmaceutical preparation 
of the gene delivery system can be introduced systemically, e.g., by 
intravenous injection, and specific transduction of the fir, protein in 
the target cells occurs predominantly from specificity of transfection 
provided by the gene delivery vehicle, cell-type or tissue-type expression 
due to the transcriptional regulatory sequences controlling expression of 
the receptor gene, or a combination thereof. In other embodiments, initial 
delivery of the recombinant gene is more limited with introduction into 
the animal being quite localized. For example, the gene delivery vehicle 
can be introduced by catheter (see U.S. Pat. No. 5,328,470) or by 
stereotactic injection (e.g., Chen et al. (1994) PNAS 91: 3054-3057). A 
therapeutic gene, such as a gene encoding an antisense RNA or a ribozyme 
can be delivered in a gene therapy construct by electroporation using 
techniques described, for example, by Dev et al. ((1994) Cancer Treat Rev 
20:105-115). 
A gene therapy preparation can consist essentially of a gene delivery 
system in an acceptable diluent, or can comprise a slow release matrix in 
which the gene delivery vehicle or compound is imbedded. Alternatively, 
where the complete gene delivery system can be produced intact from 
recombinant cells, e.g., retroviral vectors, the pharmaceutical 
preparation can comprise one or more cells which produce the gene delivery 
system. 
The compositions may, if desired, be presented in a pack or dispenser 
device which may contain one or more unit dosage forms containing the 
active ingredient. The pack may for example comprise metal or plastic 
foil, such as a blister pack. The pack or dispenser device may be 
accompanied by instructions for administration. 
The present invention is further illustrated by the following examples 
which should not be construed as limiting in any way. The contents of all 
cited references (including literature references, issued patents, 
published patent applications as cited throughout this application are 
hereby expressly incorporated by reference. The practice of the present 
invention will employ, unless otherwise indicated, conventional techniques 
of cell biology, cell culture, molecular biology, transgenic biology, 
microbiology, recombinant DNA, and immunology, which are within the skill 
of the art. Such techniques are explained fully in the literature. See, 
for example, Molecular Cloning A Laboratory Manual, 2.sup.nd Ed., ed. by 
Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 
1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); 
Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. 
No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins 
eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins 
eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 
1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A 
Practical Guide To Molecular Cloning (1984); the treatise, Methods In 
Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For 
Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring 
Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. 
eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and 
Walker, eds., Academic Press, London, 1987); Handbook Of Experimental 
Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); 
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold 
Spring Harbor, N.Y., 1986). 
EXAMPLES 
Example 1 
Preparation of an HIV-1 transgenic rat 
A transgenic rat containing a proviral HIV-1 DNA, i.e., plasmid 
pNL4-3:d1443, was prepared as follows. 
The HIV-1 proviral DNA in the plasmid pNL4-3:d1443 (represented in FIG. 1) 
is an artificial recombination of two strains. The 5' half of pNL4-3 is 
the viral isolate NY5 and the 3' half is the viral isolate LAV-1; the 
recombination splice occurred at the EcoRI site at nucleotide 5743 base 1 
being the most 5' nucleotide of the LTR). The DNA used for the 
construction of the transgenic rat included a deletion of sequence between 
the BalI and SphI sites (nucleotides 1443-4551), thereby deleting the gag 
and pol genes. The construct included both the 5' and 3' LTRs and open 
reading frames for the viral genes, env, tat, rev, nef, vif, vpr, and vpu. 
The upstream 5' splice acceptor site is left intact. Approximately 1 kb of 
human flanking sequence is present at both ends. Plasmid pNL4-3 has been 
described, e.g., in Leonard et al. (1988) Science 242:1665, illustrating 
the use of this plasmid for the preparation of a transgenic mouse. 
Specific pathogen-free inbred Fischer F-344/CrlBR (F344) rats, and outbred 
Sprague-Dawley (SD) rats, were purchased from Charles River Laboratories, 
Boston, Mass. Animals were maintained in accordance with institutional 
guidelines. 
Three week old Fisher 344 (F344) female rats (90-120 g) were superovulated 
according to Methods in Molecular Biology Vol. 18, Transgenesis 
Techniques, edited by David Murphy and David Carter, Humana Press pp 
253-256. Briefly, the female rats were injected intraperitoneally with 0.2 
IU/g body weight of pregnant mare serum gonadotropin (PMSG), followed 
46-48 hours later with an intraperitoneal injection of 0.2 IU/g body 
weight of human chorionic gonadotropin (HCG). Each female was then placed 
in a cage with a stud male. On the morning of the next day, a check was 
made for copulatory plugs or vaginal smears were performed to check for 
sperm in the lavage. At 12:00 that day, fertilized one-cell eggs were 
flushed from the oviducts of females exhibiting either a vaginal plug or 
sperm in vaginal lavage fluid. The method for collecting fertilized eggs 
was identical to that previously described for mice (Hogan et al. 1994 and 
Murphy et al. 1993). Two types of culture media were used for in vitro 
manipulations of rat embryos. M16 was used for maintaining the eggs at 
37.degree. C. gassed with 5% CO.sub.2. M2 media was used for in vitro 
manipulations outside the CO.sub.2 incubator for periods less than 30 
minutes. Eggs were collected from the ampulla in M2 media containing 300 
migcrograms/ml of hyaluronidase. Following removal of the cumulus cells, 
eggs were washed twice in fresh M2 and transferred to CO.sub.2 
equilibrated M16 and incubated at 37.degree. C. until required for 
microinjection. 
Pseudopregnant rats were obtained as follows. Female SD rats, at least 8 
weeks of age, were maintained on a 12 hour day and 12 hour night cycle so 
that they ovulate and mate every 4 days. The stage of estrous was 
determined prior to placing them with vasectomized rats. Sexually mature 
SD females were anasthetized with Methozyflurane, their vagina flushed and 
the contents dried and stained with a modified Wright's stain (e.g., 
Dip-Quick). The vaginal contents were examined at 40.times. magnification 
and each female was staged as to their position in the estrous cycle. SD 
females found to be proestrous were placed with vasectomized SD males, on 
day 0 by 18:00 hr to generate pseudopregnant recipients. 
Vasectomized male rats were obtained by a surgical procedure (Hogan et al., 
supra). Male rates were anasthetized with 60 mg/kg Ketamine and 7.5 mg/kg 
Xylazine. The abdomen was shaved and cleaned and the body wall incised and 
the left and right vas deferens were cauterized. The body wall was then 
closed with wound clips and the rat caged individually in a warm place 
until recovered. 
Microinjection of Fisher 344 X Sprague Dawley eggs and transfer to day one 
pseudopregnant Sprague-Dawley females were carried out as follows. For 
injection, the eggs were transferred to M2 medium. Eggs were sequentially 
held in place by a blunt pipet (outside diameter about 100 p) while the 
tip of the injector pipet was inserted through the zona pellucida and 
vitellus and into one of the pronuclei. The DNA solution consisting of 
plasmid pNL4-3: d1443 at a concentration of 2 ng/ml in the injector pipet 
was slowly discharged by using a 100 .mu.l Hamilton syringe connected to a 
micrometer. The injector pipet was filled with silicone oil except for the 
DNA solution. After injection, the eggs were transferred to the oviducts 
of pseudopregnant Sprague-Dawley female rats. The procedure was identical 
to that described for mice (E. Lacy et al., Manipulating the Mouse Embryo, 
Cold Spring Harbor Press, N.Y. 1994; and Methods in Molecular Biology Vol. 
18, Transgenesis Techniques, edited by David Murphy and David Carter, 
Humana Press). Briefly, the recipient was anesthetized as previously 
described and the oviducts exteriorized by a surgical incision made at the 
level of the paralumbar fossa. Approximately 15-30 embryos were 
transferred per recipient. The body wall was then closed and the recipient 
was kept warm until recovery. 
Potential founder transgenic rats were initially identified by PCR and/or 
by restriction enzyme digestion and Southern blot analysis. DNA for PCR or 
Southern blot analysis was obtained from 2-3 weeks old rat tail tips as 
per modification of the procedure of Hogan et al. (E. Lacy et al., 
Manipulating the Mouse Embryo, Cold Spring Harbor Press, N.Y. 1994). 
Approximately, 1 cm long rat tail tips were excised with a sterile scalpel 
following anesthesia with 0.02ml SQ of Lidocaine-HCL. Bleeding was 
controlled with silver nitrate. Following tail tip amputations, rats 
received Phenylbutazone 50 mg/kg, intraperitoneally as needed for pain. A 
Quiagen kit was used to extract DNA from tail tips. 
Identification and quantitation of transgenes was determined in the founder 
animals and their progeny by Southern blot analysis of genomic DNA first 
amplified by PCR Two primers (SK68 (5' AGC AGC AGG AAG CAC TAT GG; SEQ ID 
NO: 4) and SK69 (5.degree. CCA GAC TGT GAG TTG CAA CAG; SEQ ID NO: 5) were 
used to specifically amplify a 141 bp region from HIV-1 env by 
PCR-Southern blot hybridization with a .sup.32 p labeled 1.2 kb HindIII 
Nef cDNA fragment was used to confirm the identification of positive 
animals. Rats positive for the transgenic construct are referred to as 
"TgN(pNL43d14) FO1 MBC/IHV TG-1" rats. One female Sprague dawley x Fisher 
344/NHsd F1 rat was found to carry the HIV transgene. This founder 
produced many hemizygous offspring and brother-sister matings produced 
further offspring. 
Southern blot hybridization and PCR analysis indicates that copies of the 
proviral HIV genome are inserted in either 3-6 copies or 6-12 copies in 
the genome. It is likely that the transgene is inserted on two different 
chromosomes, resulting in transgenic animals having 3-6 copies or 6-12 
copies, depending on whether they have the two chromosomes carrying the 
transgene or only one. It is believed that the number of copies of the 
transgene correlates with the degree of certain characteristics of the 
phenotype of the animal, in particular with the degree of cataracts (light 
verus heavy cataracts). 
Example 2 
Phenotype of the HIV transgenic rat 
The female founder rat, TG-1, has cataracts in both eyes and a small red 
circular lesion at the base of the tail. TG-1 was mated with a normal 
Sprague Dawley male to produce the F.sub.1 generation. The F.sub.1 
offsprings had cataracts that varied from a high degree of opacity to a 
faint one. The cataracts were supplied with a large number of blood 
vessels (highly vascular). Transgenic animals had bilateral cataracts at 
birth. Two phenotypes could be distinguished: one phenotype which 
consisted of heavy, dark cataracts, and the other consisted of light, 
milder cataracts. 
In addition, at the time of weaning, most animals developed a focal skin 
lesion at the base of the tail. Of the 10 in the first litter three had 
red lesions at the base of the tail, all were females. Subsequent 
offsprings have also demonstrated that a few males have the red lesion. 
Mating of this phenotype produce offsprings with more severe skin lesions 
that cover the length of the tail and the offsprings were smaller, 
especially the females. Early pregnancy in these animals caused the 
lesions to disappear and return later in pregnancy and fully returned at 
partituation. The severe skin lesion phenotype were smaller, had a larger 
amount of proteins in urine, increased BUN and a higher alkaline urine. 
Skin lesions were that of psoratic dermatitis and hyperkerastotic lesions, 
mild kidney sclerotic lesions, and inflammatory lesions in the CNS. Thus, 
the HIV transgenic rat of the invention displays many of the pathology 
seen in humans with HIV, including retarded growth, CNS disturbances, mild 
to severe skin lesions, kidney problems and encephalitis. 
Since the transgenic rat has a similar phenotype to that of a transgenic 
mouse containing the pNL4-3 proviral DNA, the phenotype of the transgenic 
rat is most likely not due to an insertional inactivation event. 
Example 3 
Expression pattern of HIV in HIV transgenic rats 
Expression of HIV genes in the HIV transgenic rats obtained as described in 
Example 1 was determined by RT-PCR as follows. 
Rat tissues (i.e. eye, skin, muscle, brain, bone, heart, adrenal glands, 
kidney, large intestine, liver, lung, pancreas, small intestine, spleen, 
stomach, testicle, tongue, and thymus) were necropsied and snap-frozen in 
liquid nitrogen. The tissues were stored at -84.degree. C. until 
processing. The tissues were homogenized in approximately 1 ml of Trizol 
(life Technologies) using a PowerGen Homogenizer (Fisher Scientific). 
After homogenation the samples were incubated at room temperature for 5 
minutes to permit the complete dissociation of nucleoprotein complexes. 
0.2 ml of chloroform was added to the samples before shaking for 2-3 
minutes by hand. The milky pink samples were then centrifuged at 12,000 g 
for 15 minutes at 4.degree. C. The mixture separated into a lower, red, 
phenol-chloroform phase, an interphase, and a colorless upper aqueous 
phase, which contains the RNA. The aqueous phase was transferred to a 
fresh tube, and mixed with 0.5 ml isopropyl alcohol. The samples were 
incubated for 10 minutes at room temperature then centrifuged at 12,000 g 
for 10 minutes at 4.degree. C. The RNA precipitate formed a pellet on the 
side and bottom of the tube. The supernate was removed from the pellet. 
The pellet was then washed once with about 1 ml of 75% ethanol. The sample 
was then vortexed and centrifuged at 7,500 g for 5 minutes at 4.degree. C. 
The supernate was once again removed, and the pellet was allowed to 
air-dry for up to 30 minutes. DEPC water was then added to redissolve the 
pellet. To assist in dissolving the pellet, the samples were incubated for 
10 minutes (sometimes longer) at 60.degree. C. The samples were then 
stored at -80.degree. C. 
cDNA was prepared from the RNA as follows. 20 .mu.l of the RNA sample was 
incubated with 20 U of DNAse I (10 Units/.mu.l) for 1 hour at 37.degree. 
C. The sample was then phenol extracted (Trizol method above) to remove 
the DNA and DNAse protein from the RNA. The RNA was precipitated with 
ethanol and sodium acetate overnight. The pellet was washed, and dissolved 
as described above. 2 .mu.l of the sample was diluted with 200 .mu.l of 
deionized water. The sample was then quantitated to determine the amount 
of RNA present. 17 .mu.l was made to contain 2 .mu.g, of Dnased RNA. On 
ice, a cocktail of 1 l Random Heximers 100 .mu.M, 6 .mu.l 5.times. Reverse 
Transcriptase Buffer, 3 .mu.l DTT 0.1 M, 1.5 .mu.l DNTP 10 mM, 0.5 .mu.l 
RNAse inhibitor, and 1 .mu.l Reverse Transcriptase (200 u) Moloney Murine 
Leukemia Virus Reverse Transcriptase was added to the Dnased RNA. For 
samples reversed transcribed with the Art7 primer, 1.6 .mu.l Art7 and 5.4 
.mu.l 5.times. RT buffer was used. The mixture was incubated for 1 hour at 
37.degree. C. The samples were then heated at 95.degree. C. for 5 minutes 
to kill the enzyme. The samples were immediately put on ice. 
PCR was performed as follows. 5 .mu.of cDNA was added to 40 .mu.l of PCR 
SuperMix (Gibco). The PCR SuperMix contains 22 mM Tris-HCl (pH 8.4), 55 mM 
KCl, 1.65 mM MgCl.sub.2, 220 .mu.M dGTP, 220 .mu.M dATP, 220 .mu.M dTTP, 
220 .mu.M dCTP, 22 U recombinant Taq DNA Polymerase/ml, stabilizers. 5 
.mu.l of 5', 3' primers (20 .mu.M) were added to the reaction mixture. The 
samples were first denatured for 3 minutes at 95.degree. C. The parameters 
for PCR amplification were as follows: 35 cycles, each with denaturation 
at 95.degree. C. for 1 minute, annealing at 60.degree. C. for 2 minutes, 
and extension at 72.degree. C. for 2 minutes. The final cycle was followed 
by a 5 minute extension at 72.degree. C. The samples were then held a 
4.degree. C. 
The sequences of the primers and probes used for cDNA synthesis and 
detection as well as the expected products are as described in Bruggeman 
et al. (1994) Virology 202:940. Since the mRNA encoding all HIV-1 proteins 
are processed from the same precursor RNA with alternative splicing and 
all HIV-1 mRNAs have identical 5' exons, the 5' sense primer used for all 
cDNA synthesis was US (TAG TAG CAT GCT CTC TCG ACG CAG GAC TCG GCT TGC; 
SEQ ID NO: 1). The primer pair US and ART7 (ATG ATC TGC AGT TCT ATT CCT 
TCG GGC CTG TCG; SEQ ID NO: 3) was used to amplify the tat, rev, and nef 
genes. Probing of the amplified products with S1 identifies a 402 bp tat 
fragment, while probing with S2 identifies 402 bp-tat and 219/225 bp-rev 
fragments and probing with S3 identifies 402 bp-tat, 219/225 bp-rev and 
203 bp-nef fragments. The primer ART5 is downstream from the slice 
acceptor for vif and the primer ART2 is downstream from the initiation 
codon of Env. The primer pair ART5/US amplifies vif mRNA and generates a 
338 bp fragment when probed with S4. Primers ART2 (ACC TCC TGC AGC ACA GGT 
ACC CCC ATA ATA GAC TGT G; SEQ ID NO: 2) and US was used to amplify env 
mRNA and generated a 446 and 665 bp product when probed with the S3 probe. 
The 5' and 3' primers for G3PDH and SK68 (5' AGC AGC AGG AAG CAC TAT GG; 
SEQ ID NO: 4) and SK69 (5' CCA GAC TGT GAG TTG CAA CAG; SEQ ID NO: 5) for 
Env were used to amplify regions of cDNA generated from random hexamers. 
Following amplification, the reaction mixtures were subjected to 
electrophoresis, the nucleic acids were transferred onto a blot and the 
blot was hybridized with the following probes: 
S1 GAG CCA GTA GAT CCT AGA CTA GAG C (SEQ ID NO: 6); 
S2 CTT AGG CAT CTC CTA TGG CAG GAA (SEQ ID NO: 7); 
S3 ACC TCG CAT GCG AAG AAG CGG AGA CAG CGA CGA AG (SEQ ID NO: 8); and 
ENV TGA CGC TGA CGG TAC AGG CC (SEQ ID NO: 9). 
The results of tissue expression analysis indicate the presence of 
transcripts of about 7 kb (full length gag-pol mRNA), 4 kb (singly spliced 
env. mRNA) and 2 kb (multiply spliced tat, rev, and nef mRNA) in numerous 
tissues including the eyes, skin, and muscle and moderate expression in 
the brain and heart, and light expression in bone and bladder. No 
detectable levels of Env mRNA were found in the adrenal glands, kidney, 
large intestine, liver, lung, pancreas, small intestine, spleen, stomach, 
testicle, tongue, and thymus. 
Example 4 
gp120 is present in the serum and on the surface of PBMCs in HIV transgenic 
rats 
Expression of the envelope protein in serum and PBMC was assayed by ELISA 
capture and flow cytometry, respectively. The ELISA assay indicated that 
two of the hemizygous transgenic animals containing gp120 in their sera at 
levels of approximately 145 pg/ml. 
Multicolor flow cytometry of PBMCs was performed on a FACSCalibur 
(Becton-Dickinson Mountain View, Calif) as previously described (Taurog 
and El-Zaatari (1988) J Clin Invest 82, 987-92; Taurog et al. (1988) J 
Clin Invest 82, 987-92). Briefly, Ficoll-Hypaque purified peripheral blood 
mononuclear cells were incubated with saturating concentrations of F105 
human anti-env antibody (available from the AIDS Repository), washed, then 
incubated with anti-human-FITC secondary antibody (available from 
Pharmigen Commercial). The staining was done in the presence of 1% human 
AB serum (available from Sigma Commercial). After washing, the cells were 
fixed in 1% paraformaldehyde before analysis on a FACScan flow cytometer 
(Becton Dickinson, Mountain View, Calif). Viable lymphocytes were selected 
for analysis by gating of forward and 90.degree. light scatter. 
The FACS analysis, which is shown in FIG. 2, indicates that gp120 was 
readily detectable on the surface of the entire PBMC population. Thus, the 
data indicate that, contrary to mice transgenic for pNL4-3:d1443, the 
gp120 env protein is expressed on the surface of PBMCs of IRV transgenic 
rats and shed into their serum. 
Example 5 
Production of an HIV/human CD4 double transgenic rat 
A rat transgenic for HIV, e.g, HIV-1 and human CD4 can be prepared by 
crossing an HIV-1 transgenic rat described in the previous examples with a 
rat containing a human CD4 transgene and selecting for offspring carrying 
both transgenes. 
A human CD4 transgenic rat can be prepared as follows. A construct 
containing human CD4 transgene, such as pES/CD4 (Louis Flamand; hCD4 is 
also described in Gillespie et al. (1993) Mol. Cell. Biol. 13:2952), 
containing a 6 kb Mlu 1-Sal 1 fragment (ES/CD4) with the CD4 structural 
gene and its regulatory sequence can be linearized and used to prepare 
transgenic rats using the technique of pronuclei injection described above 
for the preparation of the HIV transgenic rat. 
The offspring is tested for the presence of the HIV transgene as described 
above and for the presence of the human CD4 transgene using the PCR 
primers that are specific for the human CD4 gene and which do not interact 
with the rat CD4 gene or by Southern blot hybridization using a probe 
hybridizing specifically to human and not to rat CD4. Expression of the 
transgene can be determined by Northern blot and/or flow cytometry or FACS 
analysis, as described above. For example, to detect hCD4 expression, PBMC 
can be resuspended at a concentration of 10.sup.7 cells per ml in cold PBS 
with 2% serum on ice. A total of 10.sup.6 cells can then be reacted with 
anti-human and anti-rat CD4 antibody conjugated with APC fluorochrome and 
subjected to analysis by flow cytometry. It has been observed previously 
that human and rat CD4 antibodies do not cross react. 
Infection of hCD4 transgenic rats can be performed as follows. Mature (6 to 
8 weeks old transgenic rats can be inoculated either intravenously (IV) or 
intraperitoneally (IP) with various concentrations of HIV (IIIB) (0.1-20 
TCID.sub.50) or with 10.sup.5 HIV-1 (IIIB)-infected CEM cells. Control 
animals can be non transgenic rats injected with non-infectious virus and 
hCD4 transgenic rats infected with the NSI HIV-1 (BA-L) or with diluted 
pellets from non-infected CEM cells. The presence of HIV-1 antibodies and 
viral antigen (p24) in the sera can then be analyzed every 2 weeks for the 
first two months and at 4 months post inoculation using a commercially 
available ELISA test. Rat PBMCs can be isolated on Ficoll-hypaque and 
1.0.times.10.sup.6 cells can be cultured with 0.3.times.10.sup.6 CEM 
cells. Simultaneously, 10.sup.6 PBMC can be treated with 3 .mu.g/ml of PHA 
overnight and then cultured with CEM. Cultures can be examined for CPE for 
1 month and supernatant can be checked for antigen production by ELISA 
weekly. 
Infection by HIV can also be tested in rats which are also trangenic for a 
construct containing the HIV-1 LTR upstream of DNA encoding the green 
fluorescent protein (GFP), for example. Infection of a cell containing 
this construct will result in stimulation of the LTR and therefore the 
GFP. 
Equivalents 
Those skilled in the art will recognize, or be able to ascertain using no 
more than routine experimentation, many equivalents of the specific 
embodiments of the invention described herein. Such equivalents are 
intended to be encompassed by the following claims. 
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# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 9 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - TAGTAGCATG CTCTCTCGAC GCAGGACTCG GCTTGC - # 
- # 36 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - ACCTCCTGCA GCACAGGTAC CCCCATAATA GACTGTG - # 
- # 37 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - ATGATCTGCA GTTCTATTCC TTCGGGCCTG TCG - # - # 
33 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - AGCAGCAGGA AGCACTATGG - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - CCAGACTGTG AGTTGCAACA G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - GAGCCAGTAG ATCCTAGACT AGAGC - # - # 
25 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- - CTTAGGCATC TCCTATGGCA GGAA - # - # 
24 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- - ACCTCGCATG CGAAGAAGCG GAGACAGCGA CGAAG - # - 
# 35 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "primer" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- - TGACGCTGAC GGTACAGGCC - # - # 
- # 20 
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