Multiplex gene regulation

A transgenic mouse offspring produced by the mating of a first transgenic mouse carrying a transresponder transgene whose expression is regulated by a viral gene product of HSV-1 and a second transgenic mouse carrying a transactivator transgene. A process for expressing a gene of interest which comprises the mating of a first transgenic mouse carrying a transresponder transgene whose expression is regulated by a viral gene product of HSV-1 and a second transgenic mouse carrying a transactivator transgene.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention concerns a Multiplex Gene Regulatory ("MGR") system 
which involves a two component process which provides a method of gene 
regulation and induction in trangenic animals. 
2. Background Information 
Heretofore there were two basic approaches used to control transgene 
expression. Heretofore either inducible promoters (Palmiter, R.D., 
Brinster, R.L., Hammer, R.E., Trumbauer, M.E., Rosenfeld, M.G., Birnberg, 
N.C. and Evans, R.M., (1982), "Dramatic Growth of Mice that Develop from 
Eggs Microinjected with Metallothionein-Growth Hormone Fusion Genes", 
Nature, 300, 611-615; Stewart, T.A., Patttengale, P.K. and Leder, P., 
(1984), "Spontaneous Mammary Adenocarcinomas in Transgenic Mice that Carry 
and Express MTV/myc Fusion Genes, Cell, 38, 627-637; Hanahan, D., (1985), 
"Heritable Formation of Pancreatic B-cell Tumors in Transgenic Mice 
Expressing Recombinant Insulin/simian Virus 40 Oncogenes", Nature, 315, 
115-122) or tissue specific promoter elements (Swift, G.H., Hammer, R.E., 
MacDonald, R.J. and Brinster, R.L., (1984), "Tissue-specific Expression of 
the Rat Pancreatic Eleastase 1 Gene in Transgenic Mice, Cell, 38, 639-646; 
Palmiter, R.D., Behringer, R.R., Quaife, C.J., Maxwell, F., Maxwell, I.H. 
and Brinster, R., (1987), "Cell Lineage Ablation in Transgenic Mice by 
Cell-specific Expression of a Toxin Gene", Cell, 50, 435-443); Overbeek, 
P.A., Chepelinsky, A.B., Khillan, J.S., Piatigorsky, J. and Westphal, H., 
(1985), "Lens Specific Expression and Developmental Regulation of the 
Bacterial Chloramphenicol Acetyltransferase Gene Driven by the Murine 
Alpha A-crystallin Promoter in Transgenic Mice", Proc. Natl. Acad. Sci., 
82, 7815-7819) have been used to regulate genes in transgenic mice. These 
are single tiered regulatory systems. Regulation by either inducible or 
tissue specific promoters results in some level of basal transgene 
expression which cannot be experimentally controlled. Regulation by tissue 
specific promoters merely directs expression to a certain tissue or organ 
and does not provide a direct means for controlling gene expression. 
Inducible promoters provide a method to manipulate the time of gene 
expressions however, this approach lacks a high degree of tissue 
specificity. In addition, inducible promoters are generally active during 
certain developmental stages. The basal activity level cannot be 
experimentally controlled. Thus these systems suffer from either lack of 
tissue specificity, endogenous basal activity (inducible promoters) or a 
lack of inducibility (tissue specific promoters). 
Recently Jaspal S. Khillan, Keith C. Deen, Shu-hua Yu, Raymond W. Sweet, 
Martin Rosenberg and Heiner Wetphal, "Gene Transactivation Medicated by 
the TAT Gene of Human Immunodeficiency Virus in Transgenic Mice", Nucleic 
Acids Research, Volume 16, Number 4, 1988, 1423-1430, have used the long 
terminal repeat (LTR) and the tat gene of the Human Immunodeficiency Virus 
(HIV) to construct a two tiered regulatory system. In their system the LTR 
is transactivated by the tat gene product. However, the LTR-CAT transgene 
was active in the thymus, eye, spleen, small intestine and liver in the 
uninduced state. The high basal activity of the HIV LTR severely limits 
the utility of the Khillan et al system. 
In addition to the transactivation of HIV, the tat transactivation of 
HTLV-1 has been introduced into mice. Transgenic mice which express the 
HTLV-1 tat gene is described in Michael Nerenberg, Steven H. Hinrichs, R. 
Kay Reynolds, George Khoury and Gilbert Jay, "The tat Gene of Human 
T-Lymphotropic Virus Type I Induces Mesenchymal Tumors in Transgenic 
Mice", Science, 237, 1324-1329, Sep. 11, 1987 and Steven H. Hinrichs, 
Michael Nerenberg, R. Kay Reynolds, George Khoury and Gilbert Jay, "A 
Transgenic Mouse Model for Human Neurofibromatosis", Science, 237, 
1340-1343, Sep. 11, 1987. These two papers point out that placing viral 
transactivator genes in mice can yield unpredictable results, and that 
only some transactivator genes will be useful for controlling gene 
expression in transgenic non-human mammals. 
Transgenic non-human mammals is the subject of U.S. Pat. No. 4,736,866 to 
Leder et al. 
To use transgenic animals for the analysis of gene function or to produce 
desirable gene products it is necessary to regulate the expression of the 
gene in a controlled and predictable manner. Ideally one would like to 
control both the time and site of expression in order to maintain the 
highest degree of experimental and marketing flexibility. The present 
invention, by utilizing a two-tiered system of control, provides the 
necessary degree of gene regulation required to obviate the aforesaid 
problem. 
DEFINITIONS 
MGR System--MGR System - Multiplex Gene Regulatory system 
TR--transresponder, transgenic non-human line which carries the gene of 
interest 
TA--transactivator, transgenic non-human line which carries the gene which 
produces the required transactivator; regulated by any promoter sequence; 
including the promoter normally associated with the gene in the TR line 
Promoter--regulatory sequence which controls gene expression 
IE--immediate early promoter from HSV-1 
TIF--trans-inducing factor or transactivator from HSV-1 
IE-CAT--a transgenic mouse line 
NFT--a transgenic mouse line 
CAT--chloramphenicol acetyltransferase gene 
NF-L--mouse neurofilament gene (also may be referred to herein as "NF") 
HSV-1--Herpes Simplex Virus Type 1 
LTR--long terminal repeat 
HIV--human immunodeficiency virus 
pIE--plasmid which contains the IE sequence 
BPV--bovine papillomavirus 
CRPV--cottontail rabbit papillomarvirus 
EBV--Epstein-Barr virus 
HPV--human papillomavirus 
EGF--epidermal growth factor 
EPO--erythropoitin 
FGF--fibroblast growth factor 
G-CSF--granulocyte colony stimulating factor 
GM-CSF--granulocyte-marcrophage colony stimulating factor 
PDGF--platelet derived growth factor 
TGF-beta--transforming growth factor-beta 
Hox--murine homeo-box complexes 1, 2 and 3 
AAT--alpha 1-antitrypsin 
AGP-A--alpha 1-acid glycoprotein 
AFP--alpha-fetal protein 
CRP--C-reactive protein 
GRP--gonadotropin-releasing hormone 
MBP--myelin basic protein 
SOD Cu/Zn--copper zinc superoxide dismutase 
VP--vassopressin 
WAP--whey acidic protein 
REN-2--renin 2 
fused two pieces of DNA which are ligated together to form a single 
continuous piece 
viral gene product--a protein from a viral gene, e.g., TIF gene 
SUMMARY OF THE INVENTION 
The present invention provides a two tiered system of gene regulation 
designed for use in non-human trangenic animals. In the first tier the 
gene of interest is directly regulated by a promoter which requires the 
presence of a transactivator for any appreciable expression. The tissue 
specificity, and the second tier of regulation in the invention, is 
provided by a TA line. The TA line carries the gene for the transactivator 
(TIF) regulated by a tissue specific promoter. The promoter for the TA 
transgene indirectly determines the site and time of expression from the 
IE regulated TR transgene. Only those animals with both the IE regulated 
TR transgene and an active TA transgene express the TR gene product. 
Because the TR gene requires the presence of a transactivator to induce 
expression, the invention provides a degree of control and allows for 
procedures which were not previously possible. 
The invention thus relates to a Multiplex Gene Regulatory (MGR) system. 
Using the MGR system permanent lines of transgenic animals can be 
established for any gene of interest. The MGR system comprises two 
transgenic animal lines, a "transresponder" and a "transactivator" line. 
The transresponder (TR) line carries the gene of interest. This gene is 
fused to and regulated by a promoter sequence which is specifically 
activated only in the presence of a transacting factor. In the absence of 
the required transacting factor, there is little or no transcription of 
the transgene in the TR line. The transactivator (TA) line carries the 
gene which produces the required transactivator. The gene for the 
transactivator can be regulated by any available promoter sequence, 
including the promoter normally associate with the gene in the transgenic 
TR line. When heterozygous TR and TA lines are mated, approximately one 
quarter of the offspring inherit both of the transgenes. Only in these 
animals is the TR gene expressed. In the MGR system expression of the gene 
of interest is regulated by the presence or absence of the transactivator. 
Thus, the temporal, spatial and tissue specific pattern of TR gene 
expression is defined by the promoter which regulates the TA gene (see 
Table 1 hereinbelow). This two tiered system of regulation provides a 
highly flexible means for regulating transgene expression. The MGR System 
incorporates both the inducible character and the tissue specificity of 
the single tiered methods currently in use, but eliminates the 
unregulatable basal activity inherent to the one tiered systems. The MGR 
system is thus useful for analyses of gene function and advantageous for 
regulating commerical transgenic products. 
The present invention concerns a transgenic non-human animal that carries 
integrated within its genome a transresponder transgene which comprises a 
gene of interest and a promoter sequence which is able to be regulated by 
a transactivator gene or a viral gene product and wherein in the absence 
of the transactivator gene or a viral gene product there is little or no 
expression of the gene of interest. 
The present invention also concerns a transgenic non-human animal that 
carries integrated within its genome a transactivator transgene which 
comprises a promoter element fused to a coding sequence of a 
transactivator gene, the transactivator gene able to induce expression of 
a transresponder transgene and wherein expression of a transactivator gene 
product is not detrimental to developing embryos or adults. 
The present invention is also directed to a transactivator gene fusion that 
comprises a promoter element fused to the coding sequence of a 
transactivator gene, the transactivator gene able to induce expression of 
a transresponder transgene, and wherein expression of a transactivator 
gene product is not detrimental to developing embryos or adults. 
Still further, the present invention relates to a non-human transgenic 
animal offspring produced by the mating of a first transgenic non-human 
animal that carries integrated with its genome a transresponder transgene, 
the transresponder transgene containing a gene of interest and a second 
transgenic non-human animal that carries integrated within its genome a 
transactivator transgene, the first and second transgenic animals being of 
the same species and of opposite sexes, the offspring carrying the 
transresponder transgene from the first transgenic animal and the 
transactivator transgene from the second transgenic animal, in the absence 
of the transactivator transgene there being little or no expression of the 
transresponder gene and the expression of a transactivator gene product 
not being detrimental to developing embryos or adults. 
The present invention also involves a process for producing a gene product 
comprising: 
a. introducing into a first non-human animal a transresponder transgene 
containing a gene of interest and a promoter sequence, 
b. introducing into a second non-human animal a transactivator transgene, 
the first and second animals being of the same species and being of 
opposite sexes, 
c. mating the first and second animals so as to produce an offspring 
carrying the transresponder transgene and the transactivator transgene, in 
the absence of the transactivator transgene there being little or no 
expression of the transresponder gene, in the presence of the 
transactivator, the transresponder gene being induced and the expression 
of the transactivator gene product not being detrimental to developing 
embryos or adults, 
d. recovering the gene product from the offspring. 
The present invention also concerns a process for producing a gene product 
comprising: 
a. introducing into a non-human animal a transresponder transgene 
containing a gene of interest and a promoter sequence which is able to be 
regulated a transactivator gene or by a viral gene product, 
b. infecting the animal with a virus, the virus being able to activate the 
transresponder transgene, and 
c. recovering the gene product from the animal. 
Viruses such as HSV-1, picornavirus, rhinovirus, hepatitis virus, 
reoviruses, arboviruses, rhabdoviruses, paramyxoviruses, orthomyxoviruses, 
togaviruses, arenaviruses, coronaviruses, bunyaviruses, parvoviruses, 
papovaviruses, poxviruses, poliomyelitis, aseptic menigitis, rabies, 
measles, vaccinia, influenza, Epstein-Barr virus, adenovirus, HIV viruses, 
cytomegalovirus and Norwalk type virus, just to mention a few, can be used 
in step b of the above described process. Included in the term virus is 
meant virus fragments that may or may not be infectious.

DETAILED DESCRIPTION OF THE INVENTION 
MGR Components 
The MGR system consists of a series of plasmid DNAs and two lines of 
transgenic mice. The plasmid DNAs are used in the construction of 
transgenic mice, and provide the essential components of the MGR system 
required for the analysis and regulation of other genes. The transgenic 
mouse (murine) lines IE-CAT and NFT use the HSV-1 IE promoter and TIF 
transactivator and have been used herein to demonstrate the MGR system, 
however, other animal lines and promoter transactivation pairs can be 
used. Table 1 hereinbelow is a non-limiting list of several known 
transactivators which can be employed in a MGR system. 
TABLE 1 
______________________________________ 
Transactivator Organism 
______________________________________ 
IE175, IE110 HSV-1 
GAL4, GCN4, HAP1 Sacchromyces cerevisaie 
BMLF-1, BMRF-1, BRLF-1 
EBV 
E2 CRPV, HPV, BPV 
X gene hepatitis B-virus 
IE1 Murine cytomegalovirus 
______________________________________ 
MGR Plasmids 
Non-limiting examples of plasmids for use in the construction and operation 
of the MGR system are pP02, pPOH14, pCA15, pCAT, pIE, pIEZ, pTIF and 
pNF-TIF. The plasmids pIE and pTIF provide the IE promoter (pIE) and a TIF 
coding sequence (pTIF). 
The plasmids pP02, pPOH14, and pCA15 have been described (O'Hare, P. and 
Hayward, G.S., (1985), "Three Trans-acting Regulatory Proteins of Herpes 
Simplex Viurs Modulate Immediate-early Gene Expression In a Pathway 
Involving Positive and Negative Feedback Regulation", J. of Virol., 56, 
723-733; O'Hare, P. and Hayward, G.S., (1987), "Comparison of Upstream 
Sequence Requirements For Positive and Negative Regulation of Herpes 
Simplex Virus Immediate-early Gene by Three Virus-encoded Trans-acting 
Factors", J. of Virol., 61, 190-199). 
The pPOH14, and pCAT plasmids provide the material used for the 
construction of the transgenic mouse lines IE-CAT. Plasmids pPOH14 and 
pCAT contain a fusion of the immediate early (IE) promoter from the ICP4 
gene of HSV-1 to the bacterial reporter gene for chloramphenicol 
acetyltransferase (CAT) along with the splice and polyadenylation signals 
from SV40. The IE regulatory element is a 330 base pair 5' fragment of 
ICP4 which includes approximately 30 base pairs of 5' untranslated 
sequences, the TATA box and 3 TIF responsive cis-regulatory sequences. The 
sequence for this promoter fragment has been previously reported (Murchie, 
M.J. and McGeoch, D.J., (1982), "DNA Sequence Analysis of an 
Immediate-early Gene Region of the Herpes Simplex Virus type I Genome (Map 
Coordinates 0.950 to 0.978)", J. Gen. Virol., 62, 1-15). 
IE-CAT gene fusion has been show in tissue culture cells to be activated by 
both HSV-1 infection and by transfection of the HSV-1 TIF gene (O'Hare and 
Hayward, 1985, supra, O'Hare and Hayward, 1987, supra). The fusion gene 
was excised from these plasmids and micro injected into single cell mouse 
embryos to produce the IE-CAT line of transgenic mice. The transgenic 
IE-CAT mouse lines are general purpose TR lines designed to test the MGR 
system. 
Plasmid pTIF contains the 1.5 kb BamHI, AsuII fragment of pCA15 subcloned 
into a modified pGEM vector (Promega). This construct contains 60 bp of 5' 
untranslated leader, the entire TIF open reading frame and the endogenous 
polyadenylation signal. The vector provides multiple restrction sites to 
facilitate the construction of TA gene fusions. The entire TIF sequence 
has be reported (Pellett, P.E., McKnight, J.L.C., Jenkins, F.J. and 
Roizman, B., (1982), "Nucleotide Sequence Of a Protein Encoded in a Small 
Herpes Simplex Virus DNA Fragment Cabable of Trans-inducing Alpha Genes", 
Pro. Natl. Acad. Sci., 82, 5870-5874). 
Plasmid pNF-TIF contains a 1.5 kilo base portion of the mouse neurofilament 
gene (NF-L) fused to the open reading frame of the HSV-1 TIF gene. The 
endogenous NF-L gene is one of a family of neuro-specific structural genes 
(Lewis, S.A. and Cowan, N.J., (1986), "Anomalous Placement of Introns in a 
Member of the Intermediate Filament Multigene Family: An Evolutionary 
Conundrum", Mol. and Cell Biol., 6, 1529-1539). The NF-L gene encodes a 
68,000 dalton protein which is expressed on day 11 of mouse development 
and throughout the adult life (Julien, J.P., Meyer, D., Flavel, D., Hurst, 
J. and Grosveld, F., (1986), "Cloning and Developmental Expression of the 
Murine Neurofilament Gene Family", Mol. Brain Res., 1, 243-250). The 1.5 
kb NF-L regulatory element contains a TATA box and an unknown amount of 5' 
untranslated leader sequences. This regulatory region is fused to the TIF 
sequences from pTIF. The 3kb EcoRI, HindIII insert of pNF-TIF was 
microinjected into fertilized mouse eggs to produce the NFT transgenic 
mouse lines. 
The plasmid pIE contains the 330 bp BamHI, Smal regulatory region of ICP4 
subcloned into a modified pGEM vector (Promega). This plasmid provides 
multiple restriction sites for easy construction of IE regulatored TR gene 
fusions. 
The pIEZ plasmid is analogous to the pCAT plasmid described above except 
that it uses the beta-galactosidase gene of Escherichia coli in place of 
CAT as a reporter gene. 
Characteristics of Transgenic Mice Carrying Both The TR and TA Transgenes 
(1) In the absence of transactivator there is little or no expression of 
the IE regulated gene in the TR line. 
(2) The TR gene is activated through mating with a TA line. 
(3) Expression of the transactivator gene product should not be detrimental 
to either the developing embryos or adults. 
Animals Or Plants That Can Be Employed 
The invention can be applied to warm or cold blooded animals and is not 
limited to rodents such as mice. Non-limiting examples of other animals 
that can be used include pigs, monkeys, goat, sheep, horses, cows, 
chickens and turkeys. Thus the MGR system can be applied, for example, to 
the development of transgenic livestock for the production of therapeutic 
gene products. The invention can be applied to lower eukaryots and those 
plants which can be regenerated from protoplast cultures. This includes 
most dicotyledonous plants and some monocotyledons such as wheat, corn and 
rice. 
Transresponder Transgene 
Basal TR gene expression 
CAT has been utilized herein as a target gene to test the MGR system. CAT 
is commonly used to assay the activity of promoter elements both in tissue 
culture and transgenic systems. This gene is advantageous because of a 
highly sensitive enzymatic assay which detects the CAT gene product 
(Gorman, C.M., Moffat, L.F. and Howard, (1982), "Recombinant Genes Which 
Express Chloramphenicol Acetyltransferase in Mammalian Cells", Mol. Cell. 
Biol., 2, 1044-1051). To determine the basal activity level of the IE-CAT 
gene fusion, CAT assays were conducted on a spectrum of newborn tissue 
samples. FIG. 2 demonstrates that there is little or no CAT activity in 
two of the transgenic TR lines (IE-CAT8 and 35). Even when a vast excess 
of protein is used in the CAT assay, there was encountered difficulty in 
detecting a significant level of CAT activity in these animals. The basal 
level of CAT activity in two other TR lines has been analyzed. All four 
lines exhibit little or no CAT activity in any of the tissues tested. 
IE promoters 
The size of the IE promoter influences the basal and induced levels of gene 
activity. A non-limiting example of a promoter that can be used in the 
invention is a 330 bp IE promoter fragment. O'Hare, P. and Hayward, G.S., 
(1987), J. of Virol., 61, 190-199 have demonstrated in tissue culture 
cells that both smaller and larger fragments form the ICP4 regulatory 
region exhibit quantitatively different levels of basal and induced 
activity. In general, larger fragments exhibit lower basal and high 
induced activity levels then smaller pieces. However, ICP4 promoter 
fragments much larger then the 330 bp element contain a second overlapping 
and divergent promoter element. The presence of this element which is 
oriented in the opposite direction form the ICP 4 promoter is probably 
disadvantageous for use in transgenic animals. The smaller IE promoter 
fragments which can be isolated from pIE may, however, be required for 
some gene fusions in order to maintain an acceptably low level of basal 
activity. It is also possible to use promoters from the other HSV 
immediate early genes. 
Transactivation 
Without a Transactivator Transgene 
To demonstrate that the IE-CAT gene present in the TR lines is still active 
adult mice were infected with HSV-1 by ocular scarification. O'Hare and 
Hayward (1985), supra had previously demonstrated that the IE-CAT gene 
fusion is strongly activated in tissue cultures cells when those cells are 
infected with HSV-1. An analogous experiment was conducted except that 
transgenic animals were used instead of tissue culture cells. FIG. 3 shows 
the results of CAT assays on eye tissue at three and seven days post 
infection. The infected transgenic animals exhibit an easily detected 
level of CAT activity. Uninfected transgenic and non-transgenic animals 
exhibit no CAT activity. This experiment demonstrates the inducible 
character of the IE-CAT transgene. Similar results were obtained with the 
IE-CAT33 and 38 TR lines. 
With a Transactivator Transgene 
A final test of the MGR system is to activate CAT in the transgenic IE-CAT 
line by mating with a TA line. For this experiment a TR line, homozygous 
for the IE-CAT8 transgene was mated to a NFT male. This male was the 
original transgenic "founder" animal which apparently contained two unique 
integration sites. Only one of these integration sites expressed the TIF 
gene product. Seven of the eleven offspring inherited a NF-TIF transgene 
(FIG. 4A). Four of these animals contained the active NF-TIF gene and all 
exhibited CAT induction in samples from the brain and spinal cord. No CAT 
activity was detected in the offspring with the inactive NF-TIF transgene 
or in the absence of a NF-TIF transgene (FIG. 4B). These experiments 
clearly demonstrate the MGR system. 
Effects of TIF expression 
Due to the large size of the mouse genome it is likely that the TIF protein 
may induce expression from some endogenous mouse genes (O'Hare, P. and 
Goding, C.R., (1988), "Herpes Simplex Virus Regulatory Elements and the 
Immunoglobin Octamer Domain Bind a Common Factor and Are Both Targets for 
Virion Transactivation", Cell, 52, 435-445). This induction could result 
in undesirable side effects such as developmental abnormalities, 
sterility, or cancers. Whether these side effects appear probably depends 
on the time and site of TIF expression. It is significant then that the 
NFT line, which is likely to express TIF throughout the central nervous 
system, breeds well and has no apparent health problems. As of the filing 
date hereof, applicants' oldest NFT animals were 9-12 months old and they 
exhibit no sign of deleterious effect from TIF expression. 
Using a different promoter, TIF expression was initiated at an early stage 
of mouse development (8.5 days) in a variety of mesodermal tissues (data 
not shown). These mice were also healthy and breed well. In contrast it 
has been attempted to make a TA line using a chick beta-actin promoter 
fused to TIF. The beta-actin promoter is a strong regulatory element which 
should be active throughout development in nearly all cell types. 
Apparently the beta-actin-TIF construct was lethal since it was not 
possible to produce any transgenic animals. This suggests that TIF 
expression beyond a certain level or in some tissue/stages cannot be 
tolerated during mouse developmental. 
Model for MGR System 
Although any number of different transactivator promoter pairs with the 
characteristics described herein can be used in the invention, an MGR 
system described herein was based on the well documented transactivation 
of Herpes Simplex Virus type 1 (HSV-1) immediate early (IE) genes 
(Hayward, G.S. and Sugden, B., (1986), "Herpesviruses" I. Genome Structure 
and Regulation. II. Latent and Oncogenic Infection by Human 
Herpesviruses", Cancer Cells 4. DNA Tumor Viruses: Control of Gene 
Expression and Replication, Butshan, M., Grodzicker, T., Sharp, P., ed., 
Vol. 4, 59-63). All of the five IE genes of HSV-1 have promoter regulatory 
sequences which are activated in trans by the HSV-1 gene product TIF 
(Mackem, S. and Roizman, B., (1982), "Structural Features of the Herpes 
Simplex Virus Alpha Genes 4, 0, and 27 Promoter-Regulatory Sequences Which 
Confer Regulation on Chimeric Thymidine Kinase Genes", J. of Virol., 44, 
939-949; Mosca, J.D., Reyes, G.R., Pitha, P.M. and Hayward, G.S., (1985), 
"Differential Activation of Hybrid Genes Containing Herpes Simplex Virus 
Immediate-early or Delayed-early Promoters After Superinfection of Stable 
DNA-Transfected Cell Lines", J. of Virol., 56, 867-878; O'Hare and 
Hayward, (1985), J. of Virol., 56, 723-733). This transactivation is 
specific for a cis- regulatory element present in one or more copies in 
each of the IE promoters. 
Transactivation by TIF requires the cis-regulatory sequences in the IE 
promoters, the TIF protein and some uncharacterized cellular products 
(Kristie, T.M. and Roizman, B., (1987), "Host Cell Proteins Bind to the 
Cis-acting Site Required for Virion-mediated Induction of Herpes Simplex 
Virus 1 Alpha Genes", Proc. Natl. Acad. Sci., 84, 71-75; Preston, C.M., 
Frame, M.C. and Campbell, M., (1988), "A Complex Formed Between Cell 
Components and an HSV Structural Polypeptide Binds to a Viral Immediate 
Early Gene Regulatory DNA Sequence", Cell, 52, 425-434; O'Hare and 
Hayward, 1987, J. of Virol., 61, 190-199). The TIF product is not a DNA 
binding protein. Instead transactivation appears to result when TIF forms 
a protein/DNA complex with one or more endogenous host factors. 
Promoters 
The MGR system of the invention makes no restrictions on the type of 
promoter used to regulate the transactivator. In FIG. 1 the murine 
neurofilament promoter was used to regulate the HSV-1 TIF gene (NF-TIF). 
Table 2 hereinbelow provides a non-limiting list of other promoters and 
their tissue specificity which are currently available and which can be 
used in the MGR process. 
TABLE 2 
______________________________________ 
Promoter Tissue 
______________________________________ 
MBP, GRH, VP Brain 
Crystallin eye 
beta-lactoglobin, WAP 
Mammary epethelium 
Protamine Spermatids 
Elastase, Insulin Pancreas 
Ren-2, CRP 
AAT, AGP-A Liver 
AFP Yolk sac 
beta-globin erythroid cells 
kIg, uIg B-cells 
M-MuLV LTR Macrophages 
collagen, vimentin connective tissue 
alpha-Actin, myosin light chain 
Muscle 
H-2(HLA), beta-2-microglobin, 
Many tissues 
SOD Cu/Zn 
Hox, Intl Developing CNS 
______________________________________ 
Modification of TIF 
The level of induction from the IE regulated transgene is influenced by a 
number of factors. The site of integration in the mouse genome and the 
number of tandemly repeated copies influence both the basal and induced 
level of gene activity. These factors are beyond control. A major 
modification of the MGR system which will enhance the level of IE 
induction involves maximizing the stability of the TIF transcripts. The 
TIF gene in pTIF contains 60 bp of 5' untranslated leader, the TIP open 
reading frame and the endogenous polyadenylation signals. This gene, as 
isolated from the HSV genome produces an unspliced mRNA. 
Brinster, R.L., Allen, J.M., Behringer, R.R., Gekinas, R.E. and Palmiter, 
R.D., (1988), "Introns Increase Transcriptional Efficiency In Transgenic 
Mice, Proc. Natl. Acad. Sci., 85, 836-840 have shown that the presence of 
one or more introns enhances the transcriptions activity of genes 
introduced into transgenic mice. By supplying an intron sequence to the 
TIF gene, plasmid pTIF-SV can be constructed by removing the endogenous 
TIF polyadenylation signals and replacing them with a splice and 
polyadenylation sequence from SV40. This modified TIF gene should, when 
fused to a promoter, exhibit higher transciptional activity and produce 
mRNA messages of greater stability. This will provide a major enhancement 
to the level of induced IE regulated gene activity. 
Characterization of TA lines 
It is important to accurately characterize the temporal and spatial 
patterns of TA gene expression. The IE-CAT TR lines of the invention are 
useful for determining the temporal pattern of transactivation in the 
offspring of a TR by TA cross, but it is difficult to localize the spatial 
patterns of CAT activity in these animals. To facilitate a spatial 
analysis of transactivation the pIEZ plasmid was constructed. This plasmid 
contains the E. coli beta-glactosidase gene fused to the IE promoter. A 
simple histochemical stain is available for visualizing beta-galactosidase 
activity in whole mounts and section of developing mouse embryos (Sanes, 
J.R., Rubenstein, J.L.R. and Nicolas, J.F., (1986), "Use of a Recombinant 
Retrovirus to Study Post-implantation Cell Lineage in Mouse Embryos", 
EMBO. 5, 3133-3142). Together these two TR lines provide a rapid and 
accurate method of characterizing TA activity. 
Other Transactivator/Promoter pairs 
The MGR system provides a high level of control for the expression of 
transgenes in transgenic animals. This level of control is achieved by 
using a transactivator/ promoter pair in a two tiered system of 
regulation. There are two fundamental criteria for this approach to work. 
The promoter should have a low basal activity level in the absence of the 
transactiator, and expression of the transactivator should not be 
deleterious to the animal. 
To augment the present system it may be possible to use the GAL4 
transactivator from Sacchromyces cerevisiae. GAL4 is a DNA binding protein 
which activates transcription in yeast by binding to the galactose 
upstream activating region or to a synthetic 17bp consensus sequence 
(Giniger, E., Varnum, S.M. and Ptashne, M., (1985), "Specific DNA Binding 
of GAL4, a Positive Regulatory Protein of Yeast", Cell, 40, 767-774). 
Using tissue culture cells two groups have demonstrated that GAL4 can 
transactivate chimeric genes containing either the yeast galactose 
upstream activating region or the synthetic 17-mer in mammalian cells 
(Kakidani, H. and Ptashne, M., (1988), "GAL4 Activates Gene Expression in 
Mammalian Cells", Cell 52, 169-178); Webster, N, Jin, J.R., Green, S., 
Hollis, M. and Chambon, P., (1988), "The Yeast UAS.sub.G is a 
Transcriptional Enhance in Human Hela Cells in The Presence of the GAL4 
Trans-activator", Cell 52, 169-178). The effects of GAL4 expression in 
transgenic mice are unknown. 
The TIF/IE based MGR system can be modified in a relatively simple way to 
incorporate GAL4 as an alternative transactivator. The synthetic 17bp GAL4 
binding site could be included in the IE promoter element. This modified 
IE element is likely to have a similarly low level of basal activity and 
should be inducible using either TIF or GAL4 as a tranactivator. If GAL4 
expression is well tolerated by the developing animal, e.g, mouse, this 
modification would extend the utility of the MGR system by permitting 
transactivation using GAL4 in tissues or at developmental stages in which 
TIF expression is lethal. In addition the inclusion of GAL4 increases the 
complexity of experiments which could be conducted using the MGR system. 
For instance, a TR line with a modified IE transgene could be mated to a 
TA animal, e.g., mouse, carrying both TIF and GAL4 controlled by different 
promoters with different tissue specificities. The offspring would have 
specific transgene induction in two different tissues when both TIF and 
GAL4 are inherited, or in each tissue spearately when only one of the 
transactivators are present. The complexity of the experiment increases in 
a similar way when the TR line carries both modified and unmodified IE 
transgenes. 
Transgenic Animal Lines and Their Uses 
Transgenic animal lines according to the invention, e.g., IC-CAT and NFT 
mouse lines, will be useful in studying infection, e.g., Herpes Simplex 
viruses, and in the development of therapeutic agents. It has been 
demonstrated that the IE-CAT line will activate CAT expression in the 
presence of infective virus. This IE-CAT line therefore can be a useful 
monitor for HSV and related virus infection. 
The IE-CAT line and other lines according to the present invention can be 
used to monitor the efficacy of experimental vaccines or other therapeutic 
agents. For instance, the IE-CAT line could be given an experimental 
vaccine and then challenged with HSV-1. The effectiveness of the vaccine 
can then be easily monitored and quantitated by simply assaying for CAT 
activity. Because the CAT assay is rapid and simple, this could reduce the 
cost of vaccine development. MGR systems according to this invention which 
utilize transactivator and promoter pairs from other viruses such as those 
listed in Table 1 could be developed for similar applications. 
The NFT line can be used for testing and development of pharmaceuticals for 
the treatment of HSV infection. The typical Herpes infection is a cyclic 
pattern of active infection, during which the virus replicates, followed 
by period of latency. At intermittent periods and for reasons which are 
not clear, the virus emerges from the latent state and initiates a new 
round of active replication. The means by which the virus goes latent are 
not clear, however, it is known that the unenveloped capsids move from the 
primary site of infection along neuronal axons towards their cell bodies 
(Cook, M.L. and Stevens, J.G., (1973), "Pathogenesis of Herpetic Neuritis 
and Ganglionitis in Mice: Evidence for Intra-axonal Transport of 
Infection", Infect Immun., 7, 272). It has been suggested that the axonal 
transport of the capsid away from the primary site of infection may limit 
the expression of viral immediate early genes by physically separating the 
DNA from the viral TIF gene product (Hayward, G.S. and Sugden, G., (1986), 
"Herpesviruses: I. Genome Structure and Regulation, II. Latent and 
Oncogenic Infection by Human Herpesviruses". In Cancer Cells, 4, DNA Tumor 
Viruses: control of gene expression and replication, Botchan, M., 
Grodzicker, T., Sharp, P. ed., Vol. 4, 59-93). If this is the case the NFT 
line which expresses TIF throughout the central nervous system should be 
unable to establish a latent infection. These animals can therefore 
provide a model system to study active HSV infection, and for the 
development of therapeutic reagents. 
Gene Products and Animal Models 
In developing the MGR system, the CAT gene was used as a reported gene. 
However, in practice, the CAT gene would normally be replaced with some 
other gene of interest. Because of the low level of IE promoter activity 
in the uninduced state, the MGR system can be used to regulate a wide 
diversity of genes including those which might adversely effect 
development. This capability renders the MGR system ideal for producing 
therapeutic gene products or for developing animal models to human 
diseases. Table 3 provides a non-limiting list of genes which could be 
used for these purposes. 
TABLE 3 
______________________________________ 
Gene products 
GM-CSF 
FGF 
TGF-beta 
EGF 
Interleukin 1, 2 and 6 
Tumor necrosis factor 
PDGF 
EPO 
Animal Models Disease 
______________________________________ 
oncogenes various cancers 
Factor VIII:C Hemophelia A 
Factor IX Hemophelia B 
Collagen I Osteogenesis imperfecta 
beta-globin beta-Thalassemia, Sickle Cell 
anemaia 
T-cell receptor alpha 
Ataxia telangiectasia 
Retinoic acid receptor, retinoic 
Developmental-abnormalties 
acid binding protein, Hox, Int-1 
G-CSF promyelocytic leukemia 
______________________________________ 
Unlike the use of inducible or tissue specific promoter regulatory systems, 
the MGR system requires the presence of two transgenes in the same animal 
to express the product. This is an important characteristic for potential 
manufacturers of transgenic livestock, since it provides a method to 
control the distribution of their transgenic product. To maintain a supply 
of animals, without buying more from the manufacturer, the purchaser would 
have to screen the DNA of the offspring using a Southern blot 
hybridization to track the TR and TA transgenes. This technique is 
reasonably sophisticated and not generally available to agriculturalists. 
It is believed that the MGR system is the only method which provides this 
capability. 
Other lines according to the invention which use different promoter 
transactivator pairs to control the MGR system may also be used to monitor 
other infectious agents (see Table 3 herein). 
Analyis of Development 
The MGR system permits some experiments which were not previously possible 
using the current regulatory methods described above. Many of the genes 
which are thought to regulate development in mannals are expressed in 
specific spatial patterns during development. These genes are thought to 
encode proteins which regulate the expression of other developmental genes 
including other regulatory genes. 
It is believed that disruptions in the pattern or changes in the level of 
expression for these genes will produce serious and possibly lethal 
developmental affects. Since natural mutations are not available, it is 
desirable to use transgenic mice to determine their developmental 
functions. To begin such an analysis one would like to answer some simple 
questions. What is the effect of expressing the gene in an inappropriate 
pattern? What is the effect of over expressing the gene in the normal 
pattern? Neither inducible promoters or tissue specific promoters would be 
of much help in this problem since they either lack the tissue 
specificity, or inducible characteristics required to control a 
potentially lethal gene. The MGR system however was designed to address 
this problem. 
First the regulatory gene to be studied is split into two portions, the 5' 
regulatory sequences and the coding region. A TR line is made using the IE 
promoter to regulate the coding region, and a TA line is made using the 5' 
regulatory sequences to control TIF expression. By mating these two lines 
one can induce transgene expression in the normal spatial pattern, but now 
at quantitative levels which are determined by the degree of 
transactivation. By making a second TA line with a different tissue 
specificity one can target expression of the TR transgene to ectopic 
sites. 
Environmental Safety Aspect 
The MGR system, because it requires two genes to activate the gene of 
interest, provides an additional level of control with respect to 
environmental safety precautions. For instance, transgenic mice might be 
made which express a gene product that may be potentially dangerous to 
humans. Using the heretofore single tiered regulatory method, such an 
animal would represent an environmental safety concern, since it is 
possible that this transgenic animal may escape from the laboratory and 
pass the transgene into the animal population. While this scenerio is 
unlikely, because of laboratory containment systems and since laboratory 
animals do not exist or breed very well in the wild, it does represent a 
serious concern to our society. The MGR system of the invention thus 
provides an additional level of safety. Only the animal, e.g., mouse, with 
both the TA and TR genes would express the potentially dangerous gene 
product. If that animal escaped, and if it could breed with wild mice, 
most of the offspring would inherit only the TA or TR genes. Only about 
one in four would inherit both the TR and TA genes. 
Detailed Description of Some of the Figures 
FIG. 1 is representation of the MGR system. The TR line contains a fusion 
transgene regulated by the HSV-1 IE promoter from ICP4. In this 
representation the TR gene is the reporter gene CAT. When using the MGR 
process the CAT gene would normally be replaced with the gene of interest 
such as those listed in Table 3. The TR transgene is inactive in the TR 
line. The TA line carries the HSV-1 TIF gene regulated by a tissue 
specific promoter element. In this representation mouse neurofilament gene 
was used to control TIF expression in the TA line. In the MGR system, 
however, any promoter element (see Table 2) may be substituted to control 
TIF expression in the TA line. When the TR and TA lines are mated the 
offspring represent either the parental genotypes, inheriting either the 
TA or the TR transgene, inherit neither transgene, or inherit both the TR 
and TA transgenes (highlighted box). In the offspring which inherit both 
transgenes the transactivator TIF is expressed in a tissue specific 
pattern defined by the promoter in the TA transgene. In those tissues 
where the TIF gene product is present, the TIF product complexes with 
other cellular transcription factors and activates the IE promoter leading 
to CAT expression. In every other tissue of the mouse where no TIF product 
is present there is no expression from the TR transgene. In this 
representation the HSV-1 IE promoter and the TIF transactivator was used 
to control the MGR process. Other promoter transactivator pairs could be 
used for an MGR system (see Table 1), however, it is essential that there 
is little or no activity of the TR transgene in the absence of the 
transactivator and that expression of the transactivator not be 
detrimental to the developing embryo or adult animal. 
FIG. 2 depicts basal CAT activity in TR lines IE-CAT8 and IE-CAT35. CAT 
assays were performed on newborn tissue samples by the method of Gorman et 
al (1982), supra. The positive control was a protein extract from mouse L 
cells transfected with pCAT. A 100 fold excess of protein was used in the 
tissue assays. No CAT activity was detected in the IE-CAT35 line. The 
liver and lung tissues of the IE-CAT8 line display a marginally detectable 
level of CAT activity. All other tissues in the IE-CAT8 line are negative. 
Abbreviations: heart (H), liver (Lv), spleen (Sp), kidney (K), lung (Lg), 
skin (Sk), and brain (B). 
FIG. 3 depicts CAT induction by HSV-1 infection. Adult TR mice of the 
IE-CAT8 and IE-CAT35 lines were anesthetized with 6mg/100g Nembutal, 
infected by ocular scarification and direct intercranial injection of 
HSV-1. Samples from both left and right eyes were examined for CAT 
activity at 3 and 7 days post infection. Both TR lines exhibit CAT 
induction by 3 days. There is no significant basal CAT activity in the 
uninfected TR animals (-) or in the non-transgenic CD1 controls. The 
positive control is protein extracted from mouse L cells transfected with 
pCAT. 
FIG. 4 depicts transactivation of IE-CAT8 mice by the NFT4 TA line. The 
NFT4 founder male was mated to an IE-CAT8 female which was homozygous for 
the IE-CAT transgene. FIG. A depicts Southern blot hybridization analysis 
of the eleven offspring. The DNA was digested with Pvu2 and the NF-TIF 
gene was detected using a radiolabelled probe to the NF-L promoter region. 
This probe detects both the NF-TIF transgene and a high molecular weight 
band which is derived from the endogenous NF-L gene. The NFT4 founder 
mouse contains two separate integration sites. One integration site 
produces a doublet near 3kb (offsprings 1, 6 and 8), while the second 
integration site has only a single band of approximately 3.3kb (offsprings 
2,4,7, and 9). FIG. 4B depicts CAT assays on brain (left) and spinal cord 
(right) samples from each offspring. Offsprings 2,4,7, and 9 which 
inherited the 3.3kb NF-TIF integration site exhibit positive induction of 
CAT activity. All other integration sites are inactive. The positive 
control is a protein extract of mouse L cells transfected with pCAT. 
The invention will now be described with reference to the following 
non-limiting examples. 
EXAMPLES 
Example 1: Plasmid isolation 
A single colony of bacteria was inoculated into 250 ml of LB media 
containing 50 .mu.g/ml ampicillin. The cells were grown overnight at 
37.degree. C. on a shaker then pelleted by spinning at 5,000 g at 
4.degree. C. for 15 minutes in a centrifuge. The pellet was thoroughly 
resuspended in 5 ml of 25 mM Tris-HCl, pH 8.0, 10 mM EDTA, 50 mM glucose, 
and 2 mg/ml lysozyme, and incubated on ice for 10 minutes. To this 
suspension 10 ml of freshly prepared 0.2M NaOH, 1% SDS was added, mixed by 
inversion and incubated on ice for 10 minutes. Then 7.5 ml of 3M potassium 
acetate pH 4.8 was added, mixed by inversion and incubated for 20 minutes 
at 37.degree. C. The supernatant was transferred to a fresh tube and 50 
.mu.l RNase (1 mg/ml) was added and incubated for 20 minutes at 37.degree. 
C. This solution was extracted twice with an equal volume of 
phenol:chloroform (1:1) and the DNA precipitated by adding 2 volumes of 
ethanol. The DNA pellet was recovered by centrifugation at 9,500 g for 30 
minutes. The DNA pellet was resuspended in 30 ml of water, and 28.5 g of 
CsCl and 1 ml of ethidium bromide (10 mg/ml) was added. This solution was 
centrifuged in a VTi50 rotor at 49,000 rpm for 16 hours. The plasmid DNA 
was collected from the CsCl gradient using a 5 ml syringe. The DNA was 
extracted 3 to 4 times with an equal volume of isoamyl alcohol to remove 
the ethidium bromide. The DNA was dialyzed overnight against 2 liters of 
TE (pH7.5) and concentrated by ethanol precipitation. 
Example 2: Plasmids and cloning 
Partial restriction maps of plasmids pCA15, pPO2, and pPOH14 are depicted 
in FIG. 5. These plasmids and their construction have been previously 
described (O'Hare and Hayward 1985, supra). To make the plasmid pCAT, the 
2.0 kb ClaI, SalI fragment of pP02 was isolated, end filled using Klenow 
and subcloned into the HincII site of pGEM2(Promega, Madison, Wisconsin, 
U.S.A.). This cloning process and a map of pCAT is presented in FIG. 6. 
The pTIF plasmid was made by subcloning the 1.7 kb BamHI, PstI fragment of 
pCA15 into the BamHI, PstI sites of pGEM2. The resulting plasmid was then 
digested with PstI and AsuII, end filed with T4 polymerase (IBI 
(International Biotechnologies, Inc.), New Haven, CT, U.S.A.) and 
religated, to produce pTIF. This cloning strategy is depicted in FIG. 7. 
The pNF-TIF plasmid was made by subcloning the 1.5 kb HindIII, SmaI 
fragment of pNF68 and blunt ligating it into the BamHI site of pTIF. This 
cloning strategy is depicted in FIG. 8. The pIE plasmid was made by 
subcloning the 330 bp SmaI, BamHI fragment from pPOH14 into the EcoRV, 
BamHI sites of a modified pGEM vector (FIG. 9). This modified pGEM is 
simply pGEM2 with the Blue script multiple cloning site. 
In all of the cloning operations the gene fragments and vector sequences 
were gel purified from 0.8-1.0% nondenaturing agarose gels and 
concentrated using elutip columns following the manufacturer's recommended 
procedure. Vectors used in the cloning procedures were cut with 
restriction enzymes (IBI and Promega), dephosphorylated with 
calf-intestinal alkaline phosphatase (Boehringer Mannheim Biochemcial, 
Indianapolis, Inc., U.S.A.) and gel purified. For blunt end ligations the 
vector sequences were treated as described in Molecular Cloning. A 
laboratory Manual, Maniatis, T., Fritsch, E.F. and Sambrook, J., (1982), 
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. For blunt ligations 5' 
overhand were end filled with Klenow. In this reaction 1 .mu.g of DNA in 
50mM Tris (pH 7.2), 10 mM MgS.sub.4, 0.1 mM DTT was mixed with 2nmoles of 
each dNTP, 1 unit of Klenow in a final volume of 20 .mu.l. The reaction 
was incubated at room temperature of 30 minutes. The DNA was recovered 
either by gel purification or ethanol precipitation. For the conversion of 
3' overhangs to blunt ends, T4 polymerase was used. In this reaction 1 
.mu.g of DNA in 33mM Tris-acetate (pH 7.9), 66mM potassium acetate, 10mM 
magnesium acetate, 0.5mM DTT was mixed with 1 .mu.l of each dNTP (2mM) and 
2 unites of T4 polymerase. The reaction (20 .mu.l) was incubated at 
37.degree. C. for 5 minutes. The DNA was recovered as described above. All 
ligations were conducted at 3:1 molar ratios of insert:vector with T4 
ligase. Ligation reactions (50 ng) were transformed into competent DH5 
cells and plated on LB agar containing 50 .mu.g/ml ampicillin. 
Example 3: Transgenic mice 
The transgenic IE-CAT and NFT mice were made by the following procedure of 
Jon W. Gordon, George A. Scangos, Diane J. Plotkin, James A. Barbosa and 
Frank H. Ruddle, "Genetic Transformation of Purified DNA", Proc. Natl. 
Acad. Sci. USA, 77, 7380-7384, (1980): All mice were maintained on a 14:10 
light-dark schedule (lights of at 10 p.m., on at 8 a.m.). Six-week-old 
females were induced to superovulate with 5 international units of 
pregnant mares' serum (Gestyl, Organon) at 4 p.m. followed 48 hours later 
by 2.5 international units of human chorionic gonadotropin (Pregnyl, 
Organon) and placed immediately with males for mating. B6D2F.sub.1 female 
mice were mated with CD-1 male mice; CD-1 females were mated with 
B6D2F.sub.1 males. On the same evening other mature CD-1 female mice were 
placed with vasectomized CD-1 male mice. On the morning after mating (day 
0) all female mice were examined for vaginal plugs. Six-week-old females 
were killed at 2 p.m. on day 0 and their oviducts were removed into 
Krebs-Ringer bicarbonate-buffered medium supplemented with bovine serum 
albumin and hyaluronidase at 1 mg/ml. Oviducts were opened with forceps 
and the fertilized eggs with remaining follicle cells were expressed into 
the dish. After 1-2 minutes, eggs were removed and washed three times in 2 
ml of culture medium equilibrated with 5% CO.sub.2 in air at 37 .degree. 
C. Eggs containing pronuclei were identified under the dissecting 
microscope and placed in lots of 20 in a microdrop of equilibrated medium, 
which was placed in a 100-mm tissue culture dish and covered with mineral 
oil (Mallinckrodt 6358). Eggs were stored in this manner in the incubator 
until microinjected. 
Microneedles were pulled from thin-walled no. 1211L Omega Dot tubing (Glass 
Co. of America) on a DK1 model 700.degree. C. pipette puller. Holding 
pipettes were pulled by hand on a microburner from G-12 capillary tubing 
(Thomas), and fire polished on a Sensaur microforge. The tips of the 
microneedles were allowed to fill with plasmid suspension by capillary 
action and the barrels were then filled with Fluorinert (3M FC77). They 
were then secured in PE-190 intramedic tubing on a Leitz 
micromanipulator. Holding pipettes were also filled with Fluorinert and 
similarly secured in PE-90 tubing. The tubing was likewise filled with 
Fluorinert and attached to 1-cm.sup.3 Hamilton syringes All manipulations 
were carried out on a Leitz microscope. 
Tissue culture dishes containing the fertilized eggs were placed on the 
microscope and eggs were positioned by holding the pipette such that a 
pronucleus near the plasma membrane was close to the microneedle. The 
microneedle was inserted into the pronucleus and a solution of the 2 kb 
ClaI and SalI of pPOH14 (IE CAT) in the 3 kb EcoRI Hind III fragment of 
pNF-TIF (NFT) was injected to cause an approximate doubling of the 
pronuclear volume (approximately 1 pl). Eggs that survived microinjection 
were removed and stored in a 30-mm tissue culture dish containing 2 ml of 
equilibrated medium until all microinjections were completed. Injection of 
40-60 embryos required 1-2 hours. 
Plugged pseudopregnant CD-1 female mice were anesthetized with Nembutal at 
6 mg/100 g of body weight. Ovaries were located through a dorsal incision. 
The ovarian bursa was torn away with no. 5 Dumont watchmaker's forceps, 
taking care not to rupture large blood vessels. The ostium of the oviduct 
was visualized under the dissecting microscope and a pipette containing 
10-20 microinjected embryos was inserted into it. The eggs were expelled 
into the oviduct and the would was closed with wound clips. Three weeks 
after the offspring were born 2cm tail samples were taken and used for 
Southern blot hybridization to identifiy the transgenic offspring. 
Example 4: DNA isolation 
High molecular weight mouse DNA was isolated from 2 cm tail samples (adult 
mice) or from skin samples (newborn mice) using the following protocol. 
The tissue sample was placed in 700 .mu.l of 50 mM Tris-HCL pH 8.0, 100 mM 
EDTA pH 8.0, 100 mM NaCl and 1% SDS. To this 35 .mu.l of Proteinase K (10 
mg/ml) was added and incubated at 50.degree. C. overnight. The sample was 
extracted twice with 700 .mu.l of phenol:chloroform and once with 700 
.mu.l of chloroform. The DNA was precipitated at room temperature with 2 
volumes of ethanol, and recovered by using a sealed microcapillary tube to 
remove the DNA. The DNA was resuspended in 100 .mu.l of water and 
quantified using a fluorimeter (Hoeffer). 
Example 5: Probes and Southern Blot Hybridization 
To identify transgenic offspring, tail DNA was analyzed by Southern blot 
hybridization. For each offspring, 15 .mu.g of genomic DNA was digest 
overnight at 37.degree. C. with PvuII (NFT) or BamHI (IE-CAT). The 
disgested DNA was fractionated on a 0.8% agarose gel, denatured in 1.5 M 
NaCl, 0.5M NaOH for 1 hour, neutralized in 1M Tris-HCl (pH 8.0), 1.5 M 
NaCl and blotted overnight on to nitrocellulose paper by capillary action. 
The nitrocellulose filter was baked at 80.degree. C. for 1-2 hours prior 
to hybridization. 
Either a 370 bp PstI fragment from the 1.5 kb BamHI, SmaI NF promoter or a 
570 kb Kpnl SacI fragment of TIF was used as a probe to identify 
transgenic TA offspring. Similarly, the 280 bp BamHI EcoRI fragment of CAT 
was used as a probe to detect the IE-CAT line. All three fragments were 
subcloned into M13mp19 and a single strand radio labelled DNA probe was 
made by primer extension. To make the probe 1 .mu.g of template DNA in 7mM 
Tris (pH 7.5), 7mM MgCl.sub.2, 50 mM NaCl, 1mM DTT was mixed with 3ng M13 
primer in a final volume of 10.mu.l. The primer was annealed to the 
template by incubating the mixture at 65.degree. C. for 2 minutes followed 
by a slow cooling to 30.degree. C. To the annealed mixture 1 .mu.l 0.1M 
DTT, 2 .mu.l of cold dNTPs (a 1:1 mixture of 2mM dGTP and DTTP), 3 .mu.l 
each of .sup.32 P dCTP and dATP (800 Ci/mmol) and 1 .mu.l of Sequenase was 
added and incubated at 37.degree. C. for 30 minutes. After extension of 
the primer 2 .mu.l of cold dNTPs (1:1:1:1 of 2mM dATP, dCTP, dTTP and 
dGTP) was added and incubated for 20 minutes. The salt concentration was 
then adjusted by adding 1 .mu.l of 833mM NaCl and the DNA was cut with 
HindIII for 1-2 hours at 37.degree. C. The single stranded probe was 
fractionated on a 4% polyacrylamide 8M urea gel, and the radio-labelled 
probe localized by exposing a piece of "KODAK XAR" film to the gel. The 
labelled probe was then cut out of the gel and the DNA electroeluted into 
a dialysis membrane. 
The nitrocellulose filter was prehybridized for 2 hours at 45.degree. C. in 
40% formamide, 4X SSC, 10% dextran sulfate, IX Denhardht's, 0.05% SDS and 
10 mM Tris-HCL (pH 7.5). Using fresh prehybridization buffer, the filter 
was hybridized for 16 hours at 45.degree. C. with 200,000 cpm/ml of single 
stranded probe. Finally the filter was washed 3 times in 3X SSC, 0.1% SDS 
at 45.degree. for 5 minutes and exposed to XAR film at -70.degree. C. 
overnight. 
Example 6: CAT assays 
CAT assays were preformed on newborn tissue samples as described by Gorman 
et al (1982) supra. Tissue samples were removed from newborn animals, 
placed in 100 .mu.l 0.25M Tris-HCl (pH 7.5) and frozen on dry ice. To 
extract the proteins the tissue samples were processed through 3 freeze 
thaw cycles with vigorous votexing and grinding of the tissue between each 
cycle. After the extraction, the cellular debris was removed by a 4 minute 
centrifugation in an Eppendorf microfuge. The supernatant was then assayed 
for CAT activity exactly as described by Gorman et al (1982), supra, as 
follows: The assay mixture contained (in a final volume of 180 .mu.l) 100 
.mu.l of 0.25 M Tris-hydrochloride (pH 7.5), 20 .mu.l of cell extract, 1 
.mu.Ci of [.sup.14 C]chloramphenicol (50 .mu.Ci/mmol; New England Nuclear 
Corp.), and 20 .mu.l of 4 mM acetyl coenzyme A. Controls contained CAT 
(0.01 U; F.L. Biochemicals, Inc.) instead of cell extract. All of the 
reagents except coenzyme A were preincubated together for 5 minutes at 
37.degree. C. After equilibration was reached at this temperature, the 
reaction was started by adding coenzyme A. The reaction was stopped with 1 
ml of cold ethyl acetate, which was also used to extract the 
chloramphenicol. The organic layer was dried and taken up in 30 .mu.l of 
ethyl acetate, spotted on silica gel thin-layer plates, and run with 
chloroform-methanol (95:5, ascending). The thin-layer plates were 
autoradiographed overnight at room temperature using KODAK XAR film. 
It will be appreciated that the instant specification and claims are set 
forth by way of illustration and not limitation, and that various 
modifications and changes may be made without departing from the spirit 
and scope of the present invention.