Transgenic mice which overexpress neurotrophin-3 (NT-3) and methods of use

Transgenic mice express increased levels of neurotrophin-3 (NT-3) in epithelium when their ancestors are microinjected with the NT-3 gene. The NT-3 growth factor expressing transgenic mice are useful in the study of neurodegenerative disorders of the brain such as Parkinson's syndrome and Alzheimer's disease, of the spinal cord motor neurons such as amyotrophic lateral sclerosis, and for testing drug candidates for the treatment of these diseases.

TECHNICAL FIELD 
The present invention relates to transgenic mice that express increased 
levels of neurotrophin 3 (NT-3) in the epidermis and other stratified, 
keratinized epithelium. The transgenic mice have an altered peripheral 
nervous system showing rescued NT-3 responsive neurons from a programmed 
cell death that normally occurs in development. This is evidenced by an 
increase in the number of trk C receptor expressing sensory neurons in the 
phenotype of the transgenic mouse. In addition, K14-NT-3 mice have larger 
Merkel cell (touch dome) sensory units in the skin than controls. The NT-3 
expressing transgenic mice of the present invention are valuable to 
researchers that study the role of NT-3 in the development and function of 
the central nervous system and peripheral nervous system. 
BACKGROUND ART 
Neurotrophin proteins support survival and differentiation of developing 
neurons. Target tissues such as skin produce limited amounts of 
neurotrophins during critical periods of development that act to rescue 
neurons from programmed cell death. Though target-derived influences of 
nerve growth factor (NGF) have been well documented, effects of other 
neurotrophins are less clear. 
During development of the vertebrate nervous system up to half of all 
neurons generated undergo a process of naturally occurring cell death (See 
Ard, M. D., Morest, 
D. K. (1984),; Intl. J. Dev. Neurosci. 2, 535-547; and Oppenheim, R. W. 
(1991). Ann. Rev. Neurosci. 14, 453-501). 
Neuronal death typically occurs shortly after neurons make functional 
connections within their target field. Survival during this period of 
innervation is thought to be dependent on the synthesis by the target 
tissue of a limited quantity of neurotropic substances. (See Thoenen, H. 
and Barde, Y. A. (1980), Physiol. Rev. 60, 1284-1334; Oppenheim, R. W. 
(1989), Trends in Neurosci. 12, 252-255; and Davies, A. M., Larmet, Y., 
Wright, E., Vogel, K. S. (1991), J. Cell Sci. 15, 111-116)). 
The competition for target field-derived neurotrophic factors is thought to 
serve at least two functions: to ensure that an appropriate number of 
synaptic contacts are made at the target and to eliminate inappropriate 
neuronal projections. (See Oppenheim, R. W. (1981),. In Studies in 
Developmental Biology (ed. W. M. Cowan) p 74-133. Oxford University Press; 
and Cowan, W. M., Fawcett, J. W., O'Leary, D. D. M., and Stanfield, B. B. 
(1984), Science 225, 1258-1265)). 
This concept is referred to as the neurotrophic hypothesis and predicts 
that the number of neurons in the adult could be increased if a higher 
concentration of neurotrophic factor(s) was available during the critical 
time when synaptic contacts are being established. 
The NT-3 neurotrophin appears to have a role in neuron survival and 
maintenance. NT-3 is a member of the brain derived neurotrophic factor 
(BDNF)/nerve growth factor (NGF) neurotrophin-4 (NT-4) gene family. The 
development and maintenance of the nervous system depends on these 
proteins known as neurotrophic factors. Widespread neuronal cell death 
accompanies normal development of the central nervous system and 
peripheral nervous system, and plays a crucial role in regulating the 
number of neurons which project to a given target field (Berg, 1982, 
Neuronal Development, 397-331; Cowan et al., 1984, 225:1258-65). 
Ablation and transplantation studies of peripheral target tissues during 
development have shown that neuronal cell death results from the 
competition among neurons for limiting amounts of survival factors 
(neurotrophic factors) produced in their projection fields. These 
observations led to the identification of nerve growth factor (NGF), which 
remains by far the best characterized neurotrophic molecule (Levi, 
Montalchini, and Angeletti, 1968, Physiol. Rev., 48:534-69; Thoenen and 
Barde, 1980, Rev. 60:1284-335). 
Because NGF supports only a limited set of neuronal populations, the 
existence of additional neurotrophic factors has long been postulated 
(Varon, S. and Adler, R., 1981, Adv. Cellular Neurobiology, 2: 115-63; 
Barde et al., 1987, Prob. Brain Res., 71:185-9). While it is now clear 
that other neurotrophic factors exist, their extremely low abundance has 
impeded their molecular characterization. Nevertheless, purification of 
small amounts of two such proteins, namely brain derived neurotrophic 
factor and ciliary neurotrophic factor have recently permitted their 
cloning and sequencing (Leibrock et al., 1989, Nature, 341:149-52; Stockli 
et al., 1989, Nature, 342:21-28; and Lin et al., 1989, Science, 
246:1023-25). 
There have been numerous reports of neurotrophic factor activity in 
extracts of a great variety of tissues and in conditioned culture media of 
many different cell types. Progress in characterizing these activities and 
purifying the compounds has been hampered by the fact that such activities 
are present in extremely small amounts, the range of picograms and 
nanograms per gram of tissue. 
Nerve growth factor (NGF) is a prototypical target-derived neurotrophic 
substance that has been shown to be essential for the survival and 
differentiation of neural crest-derived sensory neurons, sympathetic 
neurons, and forebrain cholinergic neurons. (See Levi-Montalcini, R. and 
Booker B. (1960), Proc. Natl. Acad. Sci. U.S.A 46, 384-391, and Hefti, F. 
(1986) J. Neurosci. 6, 2155-2161; Williams, L. R., Varon, S., Peterson, 
G., Wictorin, K., Fischer, W., Bjorklund, A. & Gage, F. H. (1986), Proc. 
Natl. Acad. Sci. U.S.A. 83, 9231-9235). 
U.S. Pat. No. 5,180,820 to Barde et al. discloses nucleic acid sequences 
encoding brain derived neurotrophic factor of humans. U.S. Pat. No. 
5,229,500 to Barde et al. claims pharmaceutical compositions including 
purified isolated nucleic acid sequences encoding brain derived 
neurotrophic factor. 
U.S. Pat. No. 5,169,764 to Shooter et al. discloses and claims a nucleic 
acid molecule encoding a chimeric protein which has neurotrophic activity 
and consists of a chimeric protein including two neurotrophic factors 
selected from brain derived neurotrophic factor, ciliary neurotrophic 
factor, neurotrophin-3 and nerve growth factor. 
U.S. Pat. No. 5,235,043 to Collins et al. discloses and claims a method for 
folding human mature nerve growth factor recombinantly expressed in E. 
coli wherein the protein maintains substantially full biological activity. 
U.S. Pat. No. 5,218,094 to de La Valle discloses a nominal neurotrophic 
factor isolated from mammalian brain tissue. U.S. Pat. No. 5,349,056 to 
Panayotatos discloses and claims a modified human ciliary neurotrophic 
factor. U.S. Pat. No. 5,364,769 to Rosenthal et al. discloses neurotrophic 
factor-4, isolated nucleotide sequences. U.S. Pat. No. 5,210,026 to 
Kovesdi et al. discloses human MK protein which is homologous to humor- 
and heparin-binding neurotrophic factor. 
U.S. Pat. No. 5,272,063 to Chan et al. discloses and claims a method of 
producing biologically active mature human .beta.-nerve growth factor in 
insect cells. 
International Publication WO 91/03568 to Hyman et al. discloses a derived 
neurotrophic factor and claims recombinant DNA molecule comprising a 
nucleic acid sequence encoding brain derived neurotrophic factor. 
International Publication 91/03569 to Hohn discloses neurotrophin-3. The 
publication claims a recombinant DNA molecule comprising a nucleic acid 
sequence encoding neurotrophin-3 or subsequences thereof. This 
international publication is incorporated herein by reference in its 
entirety. 
Various transgenic mice have been patented. U.S. Pat. No. 4,736,866 to 
Leder et al. discloses a transgenic non-human eukaryotic animal whose germ 
cells and somatic cells contain an activated oncogene sequence introduced 
into the animal or an ancestor of the animal at an embryonic stage. The 
embryo of the mice were microinjected with approximately 500 copies of the 
RSV-S107 c-myc plasmid. The injected eggs were transferred to 
pseudopregnant foster mothers and allowed to develop to term. The plasmid 
contains a Rous Sarcoma Virus enhancer and promoter sequence. The 
resultant mice showed expression of the c-myc gene in the salivary gland, 
spleen, testes, lung, brain, and preputial gland and intestinal tissue. 
U.S. Pat. No. 5,175,383 to Leder et al. discloses a male transgenic mouse 
containing germ cells and somatic cells which contain a recombinant gene 
which is a vertebrate gene in the Int-2/FGF family which is capable of 
promoting benign prostatic hyperplasia or hypertrophy. The fusion gene 
which is injected into the mouse embryonic tissue comprises a promoter 
sequence controlling transcription of the recombinant gene such as a 
promoter derived from the mouse mammary tumor virus and cytomegalovirus. 
The recombinant gene is preferably substantially homologous with (i.e., 
greater than 50% homologous in terms of encoded amino acid sequence) a 
naturally occurring, vertebrate gene in the Int-2/FGF gene family of 
murine growth factor encoding genes or their vertebrate counterparts, 
including the murine acidic or basic fibroblast growth factor genes, the 
murine FGF-5 gene, the murine epidermal growth factor gene, the murine 
insulin-like growth factor-1 and -2 gene, the murine .alpha.-transforming 
growth factor gene with a murine hst/KS3 gene. The transgenic mice of 
Leder et al. exhibit prostate hyperplasia and give sterile offspring. 
U.S. Pat. No. 5,087,571 to Leder et al. discloses a non-human eukaryotic 
animal whose germ cells and somatic cells contain an activated oncogene. 
The embryo of the mice were microinjected with approximately 500 copies of 
the RSV-S107 c-myc plasmid. The injected eggs were transferred to 
pseudopregnant foster mothers and allowed to develop to term. The plasmid 
contains a Rous Sarcoma Virus enhancer and promoter sequence. The 
resultant mice showed expression of the c-myc gene in the salivary gland, 
spleen, testes, lung, brain, and preputial gland and intestinal tissue. 
U.S. Pat. No. 5,175,384 to Krimpenfort et al. discloses transgenic mice 
having a phenotype characterized by the substantial absence of mature 
T-lymphocytes. The mouse is produced by introducing a transgene into a 
zygote of a mouse which comprises gene fragment which encodes a T-cell 
antigen receptor polypeptide variant which is incapable of mediating 
T-cell maturation in the transgenic mouse. 
U.S. Pat. No. 5,175,385 to Wagner et al. discloses the production of a 
transgenic mouse with enhanced viral resistance which is transmissible to 
its offspring. The transgenic mouse is prepared by introduction of a gene 
encoding a human interferon having anti-viral activity into a host mouse. 
The plasmid of Wagner et al. contains a methallothionein-1 promoter and 
the genomic human beta interferon gene. When these mice were injected with 
pseudorabies virus they showed an increased resistance to the virus and 
although many mice died they died considerably later than did the control 
animals. 
Vassar et al., in the Proceedings of the National Academy of Sciences, 
Volume 86, pages 1563-1567, March 1989, disclose "Tissue-specific and 
Differentiation-specific Expression of a Human K14 Keratin Gene in 
Transgenic Mice". This publication discloses that Vassar et al. used a 
plasmid containing a K14 keratin promoter and a K14 keratin gene sequence 
tagged with a sequence from the neuropeptide substance P and determined 
the expression of the plasmid and K14 keratin tagged substance P in the 
skin of transgenic mice. 
Vassar et al. in Gene & Development, Volume 5, pages 714-727, (1991) 
disclose "Transgenic Mice Provide New Insights into the Role of 
TGF-.alpha. During Epidermal Development and Differentiation". This 
publication discloses the construction of transgenic mice using a plasmid 
which contains the K14 keratin promoter and the TGF-.alpha. gene. Vassar 
et al. disclose that the TGF-.alpha. gene belongs to the epidermal growth 
factor family of proteins and shows structural homology with the epidermal 
growth factor. These transgenic mice showed gross phenotypic abnormalities 
in their skin including flaky outer epidermal layers with stunted hair 
growth and wrinkling. The plasmid used to create this transgenic mouse 
also included human growth hormone fusion gene. 
U.S. Pat. No. 5,387,742 discloses transgenic mice which exhibit amyloid 
brain deposits characteristic of Alzheimer's disease. 
PCT Publication WO 93/00909 is directed to a method of treating 
neurotrophin-expressing tumors by administering a sequence complementary 
to at least a portion of a RNA transcript of brain-derived neurotrophic 
factor gene. 
Heretofore there has been no convenient method to investigate how 
neurotrophic factors such as NT-3, affect neuron survival and development 
of the peripheral nervous system or provide a practical model for testing 
drug candidates for the treatment of neurodegenerative disorders of the 
brain such as Parkinson's syndrome and Alzheimer's disease and assessing 
the effect of drug candidates on the central nervous system (CNS) and 
peripheral nervous system (PNS). 
The present invention overcomes the deficiency by providing transgenic mice 
which express increased levels of Neurotrophin-3 in the epidermis and 
other stratified, keratinized epithelium. The transgene DNA construct 
injected into the mouse embryos contain a human K14 keratin promoter and 
enhancer region linked to the coding sequences of mouse Neurotrophin-3 
gene. The mice can be used to study the role of NT-3 in supporting 
neuronal survival and in neurodegenerative disorders of the CNS and PNS. 
SUMMARY OF THE INVENTION 
The invention provides a transgenic mouse whose somatic and germ cells 
contain and express a gene encoding for NT-3. The gene was introduced into 
a mouse or an ancestor of the mouse at an embryonic stage. The NT-3 gene 
is operatively linked to a human K14 keratin promoter and enhancer region. 
The increased expression of the transgene was achieved by adding a region 
from the human growth hormone gene at the 3' end of the construct. The 
NT-3 in transgenic mice appears to alter the peripheral nervous system by 
rescuing NT-3 responsive neurons from a programmed cell death that 
normally occurs in development. NT-3 transgenic mice appear to have more 
nerve terminals in the skin, particularly in association with hairs and 
touch dome mechanoreceptor endings. 
The invention also provides for a mouse wherein the NT-3 gene has been 
introduced into an ancestor of said mouse at an embryonic stage by 
microinjection. In an additional embodiment, the mouse NT-3 gene is 
further operatively linked to the human growth hormone gene. 
The invention provides for a mouse which has a phenotype characterized by 
an altered peripheral nervous system showing rescued NT-3 responsive 
neurons from a programmed cell death that normally occurs in development. 
This is evidenced by an increase in the number of trk C receptor 
expressing sensory neurons in the phenotype of the transgenic mouse. In 
addition, K14-NT-3 mice have larger Merkel cell (touch dome) sensory units 
in the skin than controls. 
Advantageously, the present invention provides a method of producing the 
transgenic mouse which overexpresses NT-3 which comprises 
(a) providing a mouse NT-3 gene operatively linked to a human K14 keratin 
promoter and enhancer region functional in said mouse; 
(b) introducing said gene into a mouse embryo; 
(c) transplanting said embryo into a pseudopregnant mouse; and 
(d) allowing said embryo to develop to term. 
Furthermore, the invention provides a method of testing the efficacy of 
drugs in treating neurodegenerative disorders comprising administering 
said drug to a mouse according to the invention, and determining the 
behavioral effects and tissue changes of said drug on said mouse. 
The above and other objects of the invention will become readily apparent 
to those of skill in the relevant art from the following detailed 
description and figures, wherein only the preferred embodiments of the 
invention are shown and described, simply by way of illustration of the 
best mode of carrying out the invention. As is readily recognized the 
invention is capable of modifications within the skill of the relevant art 
without departing from the spirit and scope of the invention.

STATEMENT OF DEPOSIT 
The plasmids used to transform the mice of the present invention have been 
deposited under the terms of the Budapest Treaty with the American Type 
Culture Collection, 12301 Parklawn Dr. Rockville, Md. 20852. The K14 NT-3 
hGH plasmid in E. coli has ATCC Accession No. 69889 and was received Aug. 
16, 1995. 
During the pendency of this application, access to the deposit will be 
forwarded to one determined by the Commissioner to be entitled thereto; 
(b) all restrictions imposed by the depositor on the availability to the 
public of the deposited material will be irrevocably removed upon the 
granting of the patent, (c) the deposit will be maintained for a period of 
at least thirty years or at least five years after the most recent request 
for the furnishings of a sample of the deposited material, (d) the deposit 
will be replaced should it become necessary due to inviability, 
contamination or loss of capability to function in the manner described in 
the specification. 
DETAILED DESCRIPTION OF THE INVENTION 
To examine target-derived effects of neurotrophin-3 (NT-3), transgenic mice 
were isolated that overexpress NT-3 in the epidermis. Experiments that 
examine the neuroanatomical and neurochemical changes induced by NT-3 
expression in skin are conducted. Neurons of the trigeminal, superior 
cervical, and dorsal root ganglia are examined. Behavioral testing is 
initiated to determine whether alteration in sensory perception is induced 
by the increased neuronal innervation. Transplantation studies examine the 
usefulness of the transgenic skin in supplying NT-3 to neuronal lesion 
sites. 
It was discovered that the production of NT-3 in transgenic mice alters the 
peripheral nervous system by rescuing NT-3 responsive neurons from a 
programmed cell death that normally occurs in development. This is 
evidenced by an increase in the number of trk C receptor expressing 
sensory neurons. 
In addition, K14-NT-3 mice have larger Merkel cell (touch dome) sensory 
units in the skin than controls. These mice can be used in conjunction 
with K14-NGF and K14-BDNF transgenic mice to examine the role and possible 
use of neurotrophic compounds in controlling the development and function 
of the nervous system. 
Neurotrophins are growth factors that have profound effects on the 
development and function of the mammalian nervous system. Transgenic mice 
were isolated that express increased levels of NT-3 in the epidermis and 
other stratified, keratinized epithelium. The transgene DNA construct 
injected into mouse embryos contains the human K14 keratin promoter and 
enhancer region linked to the coding sequences of the mouse NT-3 gene 
(FIG. 1). To increase expression of the transgene, a region from the human 
growth hormone gene was added at the 3' end of the construct. Eleven 
founder mice were isolated; three lines were developed for further use. 
The results support the neurotrophic hypothesis and specifically 
demonstrate that the skin, by regulating the timing and amount of 
neurotrophin expression independent of innervation plays a central role in 
the development of the peripheral nervous system. 
Elevated levels of NT-3 in the mice of the invention were found to increase 
the size of touch dome units, the number of Merkel cells (MC) per unit, 
and their level of neuronal innervation. In addition, the number of 
myelinated and unmyelinated axons in cutaneous nerves was increased 
approximately 60% while the number of trkC expressing neurons in the 
trigeminal ganglia doubled. 
Previous studies of transgenic mice that overexpress NGF in the skin showed 
a dramatic increase in innervation density, an increase in the number of 
trkA expressing neurons, and an overall hypertrophy of the peripheral 
nervous system (1, 2). Because skin also synthesizes the neurotrophins 
NT-3, BDNF, and NT-4 during critical periods of sensory neuron development 
(3, 4, 5), we developed additional lines of neurotrophin expressing mice. 
The transgenic mice of the invention overexpress NT-3 in the epidermis. The 
expression of NT-3 by the skin suggests that it may, like NGF, support 
distinct subsets of cutaneous sensory neurons and thereby influence 
development of innervation density and sensory responsiveness (1, 6, 7, 
8). NT-3 gene expression is detectable in developing skin at embryonic day 
9.5 (E9.5), peaks at E13, and then declines to lower adult levels (9). To 
increase levels of target-derived NT-3, transgenic mice were isolated that 
overexpress NT-3 in basal keratinocytes of the epidermis using promoter 
and enhancer sequences of the human keratin K14 gene (1, 10) (FIG. 1a). 
Expression of K14 in developing epidermis is initiated by approximately 
day 14 and continues in the adult. The onset of transgene expression, 
therefore, overlaps with the normal decline of NT-3 expression in the 
skin. Heterozygote transgenic offspring from K14-NT-3 founder mice (FIG. 
1B) appeared normal. They were characterized for transgene mRNA expression 
using Northern analysis of RNA isolated from back skin (FIG. 1C) and in 
situ hybridization analysis of tail skin (FIG. 1D). Two lines (696-2 and 
694-2) that expressed the highest levels of the K14-NT-3 transcript were 
used for this study. 
Histological examination of transgenic skin showed a remarkable increase in 
the size of touch domes (FIG. 2A, B), mechanoreceptor units innervated by 
large diameter, slowly adapting type I (SAI) sensory neurons (11). Touch 
domes of transgenic mice (FIG. 2B) were nearly doubled in size compared to 
controls (FIG. 2A). In dermis beneath touch domes an increased cellularity 
was consistently observed and appears to represent Schwann cells 
associated with the innervating fibers. An increased labeling density in 
this region with an antibody that recognizes a galactocere-broside 
component of myelin protein expressed in Schwann cells (01 antibody) is 
noted. 
To examine the nature of innervation to touch dome units, sections of 
control and transgenic skin were immunolabeled using an antibody against 
protein PGP 9.5 (FIG. 2C, D), which recognizes all neuronal fiber types, 
and an antibody against neurofilament 150 (NF 150; FIG. 2E, 2F), a protein 
primarily localized to myelinated axons. An increase in the density of 
innervation to the skin and touch dome units of the transgenic mice (FIG. 
2C, 2D) appeared in large part attributable to increased projection of 
afferents to hair follicles, particularly in regions where circular 
endings were located. Given the physiologically measured decrease in 
D-hair units in skin of NT-3 (+/-) mutant mice (12), this increase shows 
that these circular endings may represent the anatomical substrate of the 
D-hair unit. The clearly larger touch dome units of transgenics were also 
innervated by an increased number of immunoreactive fibers (FIG. 2F) 
compared to controls (FIG. 2E), suggesting that the transgenic touch domes 
are enhanced functionally as well. 
SAI afferents innervating touch domes terminate on specialized 
neuroendocrine cells known as Merkel cells (MCs). MCs are located at the 
epidermal-dermal junction and are thought to act as transducers of 
mechanical deformation (13). To determine whether MC numbers were 
increased in conjunction with the increase in touch dome area and 
innervation, control and transgenic mice were injected with quinacrine 
fluorescent dye, a compound selectively concentrated in neuroendocrine 
cell types (14). Counts of quinacrine labeled control and transgenic skin 
showed a statistically significant (p&lt;0.001) increase in the number of MCs 
per touch dome in the NT-3 transgenics (FIG. 3A). 
Gene knockout and cell culture studies have shown NT-3 supports the 
survival of myelinated sensory neurons of large axonal caliber (15, 16). 
To examine whether overexpression of NT-3 affected cutaneous myelinated 
nerves, we measured the number of myelinated fibers in control and 
transgenic mice by counting cross-sectional profiles of the saphenous 
cutaneous nerve (ie. 3B). Axon counts from control (521.+-.6.8 s.e.m.) and 
transgenic 839.+-.11.8 s.e.m.) mice showed transgenic nerves to have an 
approximate 60% increase in the number of myelinated fibers ((p&lt;0.001) 
compared to controls). This increase in axon count was reflected by the 
near doubling of the transgenic saphenous nerve cross-sectional area (FIG. 
3B, right side) compared to the control nerve (FIG. 3B, left side) (34,016 
units .mu.m.sup.2 compared to 17,196 .mu.m.sup.2, p&lt;0.001). Subsequent 
counting of unmyelinated axons showed there number to also be increased by 
60%. 
The biological effects of neurotrophins are primarily mediated through the 
trk family of tyrosine protein kinases (17, 18, 19). TrkA preferentially 
binds NGF, trkB binds BDNF and NT-4, and trkC binds NT-3. Approximately 
10% of dorsal root ganglia (DRG) sensory neurons express the trkC 
receptor, 10% express the trkB receptor, whereas 40% express trkA (20, 21, 
22, 23). 
Target production of neurotrophins affects neuron survival and 
differentiation via a trk receptor mediated uptake process that 
facilitates internalization and retrograde transport to the soma (24). 
Abundant evidence supports this mechanism for the prototypical 
neurotrophin NGF and its high affinity receptor trkA. Mice lacking a 
functional NGF gene lack trkA expressing neurons (8), whereas mice that 
overexpress NGF have an increased number of trkA expressing neurons (2). 
To examine whether increased expression of NT-3 acted to increase the 
number of sensory neurons that expressed trkC, in situ hybridization was 
carried out using a radiolabeled probe to the trkC receptor mRNA (25). 
Trigeminal ganglia from the 694-2 and 696-2 transgenic lines had an 
approximate two-fold increase in the number of trkC expressing neurons 
compared to control ganglia (Table 1). Interestingly, a plot of these 
values versus animal age (FIG. 4) revealed that, for both control and 
transgenic mice, the number of the trkC positive neurons accumulated with 
age, stabilizing by approximately three weeks. This increase in trkC 
neurons may reflect a maturation and/or refinement of the sensory system 
during the early weeks of development and demonstrates the plasticity 
inherent in the peripheral sensory system. 
Studies of NT-3 and trkC gene knockout mice showed a depletion of 
proprioceptive 1A afferents as well as the muscle spindle sensory end 
organs they innervate (15, 16, 26). The disappearance of proprioceptive 
neurons cannot, however, account for the overall 50% loss of sensory 
neurons in the DRG of NT-3 (-/-) animals and suggests other sensory 
neurons are dependent on NT-3 for their development and maintenance. 
The present studies identify cutaneous touch dome complexes and their 
associated MCs as another sensory end organ responsive to NT-3 and, by 
supposition, innervated by trkC receptor expressing neurons. This 
observation is supported by the additional innervation to touch dome 
units, the increase in MCs, and the increase in trkC expressing neurons in 
the sensory ganglia (FIG. 4). 
The enlargement of touch domes and number of MCs may result from the 
enhanced innervation by SA1 afferents generated by the increased level of 
NT-3. In support of this mechanism, studies by Nurse et al., (27) showed 
that denervation of backskin of rats led to a decline in the number of 
MCs, i.e., the sensory nerve supply was required for MC maintenance. In 
addition, MCs are depleted in NT-3 (+/-) mice which lack SA1 type fibers 
(12). 
In the present model an overabundance of appropriate SA1fiber types appear 
available to contact MCs and, interestingly, this overabundance enhances 
the development of the end organ. Effects of the epidermally expressed 
NT-3 on the cells comprising the touch dome is another possible factor in 
touch dome development, perhaps acting in a synergistic manner with the 
afferent innervation. 
Table 1. Number of TrkC receptor expressing neurons in the trigeminal 
ganglion of control and K14-NT-3 transgenic mice. Positive cells were 
counted in every tenth section of serial sectioned ganglia. Transgenic and 
control ganglia were hybridized together and therefore exposed to the same 
conditions. 
TABLE 1 
______________________________________ 
Age TrkC labeled neurons 
Sample (weeks) (avg) 
______________________________________ 
Transgenic line 696-2 
5 (n = 2) 
62 
7 (n = 1) 
259 
9 (n = 2) 
283 
26 (n = 1) 
327 
Transgenic line 694-2 
6 (n = 4) 
113 
23 (n = 2) 
342 
44 (n = 1) 
294 
Controls 63 (n = 2) 
417 
5 (n = 2) 
37 
7 (n = 2) 
90 
9 (n = 1) 
116 
23 (n = 1) 
189 
26 (n = 1) 
158 
44 (n = 2) 
156 
63 (n = 1) 
214 
______________________________________ 
To correct for variation in neuron size, recursive translation analysis was 
performed on labeled neuron cell counts. The perimeter (p) of labeled 
neuronal profiles were drawn by camera lucida and the cross sectional area 
(A) of each profile calculated from a digitized image of the drawing. The 
radius (r) of each profile was approximated using r=square root of (A/p). 
The frequency histogram of the profile radii was transformed to an 
estimate of the true frequency histogram of neuronal radii with a computer 
program developed by Rose and Rohrlich (30). Summing the frequencies in 
all bins of the resulting histogram gives an estimate of cell number in 
one section of a ganglion. The recursive translation method was repeated 
on equally spaced 20 .mu.m serial sections (for example, on every n.sup.th 
section) throughout the entire ganglion. Summing the number of cells 
across all of the sampled sections and multiplying the total by n gives an 
unbiased estimate of the true number of labeled cells. n equals the number 
of mice used per age group. 
DETAILED DESCRIPTION OF DRAWINGS 
FIG. 1 shows isolation and expression of the K14-NT-3 transgene in 
transgenic mice. Mouse NT-3 cDNA was cloned by PCR amplification of DNA 
isolated from tail using primers (5'CCAGCGGGATCCGTGATGTCCATCTTGTTTTATGTG 
3' (SEQ ID NO:1) and 5'CGGTACGGATCCGATGCCAATTCATGTTCTTCCG 3' (SEQ ID 
NO:2)) that amplified the DNA sequence encoding amino acids -140 to +124 
according to the numbering of Yancopoulos et al., (28). This region 
contains the putative signal peptide (-140 to -1), the mature peptide (+1 
to +119), and the NT-3 stop codon (+120). To facilitate cloning, primers 
contained a BamH1 recognition sequence (underlined in sequence). DNA 
sequencing showed a nucleotide change in the cloned sequence that resulted 
in a conservative amino acid change (ala.fwdarw.val) at amino acid residue 
-9!. 1(A) shows the NT-3 DNA was ligated into the BamH1 site of the 
K14-hGH cassette vector (10) and purified as described in (1). The 
K14-NT-3 sequence was microinjected into pronuclei of B6.times.C3 F1 
hybrid mouse embryos and implanted into pseudopregnant females using 
standard procedures (29). 
Four founder mice (696-2, 694-2, 709-9, and 662-8) were identified by 
Southern hybridization using a random primed .sup.32 P-dCTP labeled probe 
made to the full length NT-3 cDNA. 1(B) shows founder offspring were 
screened and transgene copy estimated by film densitometry from slot blots 
on which 2 .mu.g of denatured tail DNA was bound by baking to Nytran 
membrane and hybridized with a .sup.32 p-labeled probe to the NT-3 
sequence. Copy number estimates were 8 (line 696-2), 2 (line 694-2), 5 
(line 709-9), and 3 (line 62-8). 
Because of poor reproductive yields of homozygous mice, experimental 
analyses were restricted to hemizygous transgenics. 1(C) shows transgene 
expression was measured by Northern analysis of RNA isolated from shaved 
back skin. RNA was purified using Trizol reagent (Gibco-BRL) according to 
the manufacturers protocol. 10 .mu.g of RNA was resolved on a 1.2% agarose 
formaldehyde denaturing gel, transferred to Nytran membrane, baked at 
80.degree. C., and hybridized at 65.degree. C. in the presence of 50% 
formamide to a .sup.32 P-CTP labeled riboprobe made to the full length 
NT-3 sequence. Membranes were hybridized overnight, washed, and exposed to 
X-ray film. Lane nt, nontransgenic RNA; lane 2, 662-8 RNA; land 3, 709-9 
RNA; land 3, 694-2 RNA, and land 4, 696-2 RNA. 1(D) shows In Situ 
hybridization using a .sup.35 S-labeled antisense probe made against the 
NT-3 mRNA confirmed K14-NT-3 transgene expression in basal keratinocytes 
of the epidermis (left panel, control tail skin; right panel, transgenic 
tail skin). Conditions of probe hybridization were as described in (1). 
FIG. 2 shows histological and immunocytochemical analysis of transgenic 
skin. Flank skin from control (A) and transgenic (B) mice was hematoxylin 
and eosin stained. Note increased size of touch dome (bracketed area) and 
increased cellularity beneath touch dome in transgenic sample. Innervation 
to the skin and touch domes was visualized by immunolabeling tissue 
sections using anti-PGP9.5 (Ultraclone) (FIG. 2C, 2D) and anti-NF150 
(Chemicon) (FIG. 2E, 2F) antibodies. PGP9.5 immunoreactivity was more 
extensive overall in transgenic skin (FIG. 2D) compared to control (FIG. 
2C), particularly in areas surrounding hair follicles. 
Touch domes of NT-3 mice (FIG. 2F) were larger and appeared to have greater 
innervation density than control touch domes (FIG. 2E). Immunolabeling was 
done on 30 .mu.m thick floating sections that were blocked in 5% normal 
goad serum (NGS), 2% bovine serum albumin (SA), and 0.25% triton X-100 
made in tris buffered saline for 1 h, exposed overnight at room 
temperature to diluted antibodies (anti-PGP 9.5, 1:500 dilution of 
biotinylated goat anti-rabbit secondary for 1 h followed by a strepavidin 
complex incubation (Vector Labs)). Binding was visualized using a nickel 
cobalt enhanced diaminobenzidine reaction. Scale bars A-d, 50 .mu.m; E-F, 
100 .mu.m. 
FIG. 3 shows transgene expression of NT-3 increases the number of Merkel 
cells and cutaneous myelinated nerves. Histogram 3(A) illustrating the 
number of MCs per touch dome unit shows increased numbers of MCs 
associated with the larger touch dome areas in the NT-3 transgenic skin. 
Numbers were determined for 3 transgenic and 3 controls by counting 
quinacrine hydrochloride labeled cells on full thickness skin sections 
using a microscope equipped with FITC fluorescent optics (14). Back skin 
of adult mice was depilated and 24 h later mice were injected 
intraperitoneally (15 mg/kg) with quinacrine solution made in phosphate 
buffered saline. 20 h later mice were deeply anesthetized and back skin 
removed and analyzed. 3(B) shows on the left side a cross section of 
saphenous nerve of control mouse; right side is nerve from an NT-3 
transgenic. Saphenous nerves of transgenics were nearly double in size 
compared to control nerves and had a 40% increase in the number of 
myelinated axons. Nerve samples were collected from transgenic (n=3) and 
control (n=3) mice deeply anesthetized with Avertin and perfused 
intracardially with a solution of 2% paraformaldehyde and 2% 
glutaraldehyde made in phosphate buffer. Tissues were fixed overnight, 
embedded in epon, thin sectioned, stained with toluidine blue, and 
myelinated profiles counted with the aid of a computer. Nerve counts and 
MC counts were analyzed from the same mice. 
FIG. 4 shows enhanced expression of NT-3 increases the number of trkC 
expressing sensory neurons. To identify trkC neurons in trigeminal ganglia 
in situ hybridization was performed using a .sup.35 S-labeled antisense 
probe made against rat trkC mRNA (25). Values at timepoints listed in 
Table 1 were plotted against age and show an overall increase of trkC 
neurons for both transgenic lines compared to controls and reveal a 
significant positive correlation between the number of trkC neurons and 
age for both transgenic (line 694-2, r=0.626 p&lt;0.05) and control (r=0.802; 
p&lt;0.005) mice. Control (-"box"-); line 696-2 (-"dark circle"-); line 694-2 
(-o). 
TABLE 2 
______________________________________ 
Number of trk C 
Mouse Number 
Phenotype Labeled neurons 
______________________________________ 
711-2 Nontransgenic 
272 
696-2F1-25 
Nontransgenic 
292 Average value 
696-2NT14 
Nontransgenic 
136 for nontransgenic 
696-2NT17 
Nontransgenic 
208 mice = 227 
696-2F2-6 
Transgenic 758 
694-2 Transgenic 645 Average value 
696-2F2-30 
Transgenic 515 for transgenic 
696-2F2-27 
Transgenic 649 mice = 642 
______________________________________ 
EXAMPLE 1 
Construction of K14-NT-3 transgene and introduction into mice. Transgenic 
mice that overexpress NT-3 in the skin were generated by linking a PCR 
cloned NT-3 cDNA downstream of 2 Kbp of the human D14 keratin promoter and 
enhancer sequence. NT-3 cDNA was cloned by PCR amplification of DNA 
isolated from tail using primers (5'CCAGCGGGATCCGTGATGTCCATCTTGTTTTATGTG 
3' (SEQ ID NO:1) and 5'CGGTACGGATCCGATGCCAATTCATGTTCTTCCG 3' (SEQ ID 
NO:2)) that amplified the DNA sequence encoding amino acids -140 to +124 
according to the numbering of Yancopoulos et al. This region contains the 
putative signal peptide (-140 to -1), the mature peptide (+1 to +119), and 
the NT-3 stop codon (+120). To facilitate cloning primers were generated 
to contain a BamH1 recognition sequence (underlined in primer sequence). 
Sequencing of the cloned NT-3 showed a nucleotide change that resulted in 
a conservative amino acid change (ala.fwdarw.val) in the signal peptide 
region at amino acid residue -9!. To construct the K14-NT-3-hGH transgene 
the NT-3 cDNA was ligated into the BamH1 site of the K14-hGH cassette 
vector (Vassar and Fuchs, 1991, supra). Dr. Elaine Fuchs (University of 
Chicago) provided the K14-hGH plasmid. 
K14-hGH contains 2.1 kbp of 5' upstream sequence of the human K14 keratin 
gene and a 1.8 kbp intron containing sequence from the human growth 
hormone (hGH) gene. The hGH sequence serves to upregulate expression of 
the transgene and provides a polyadenylation signal. (See Sandgren, E.P., 
Luetteke, N.C., Palmiter, R. D., Brinster, R. L. & Lee, D. C. (1990), 
"Overexpression of TGF alpha in transgenic mice: Induction of epithelial 
hyperplasia, pancreatic metaplasia, and carcinoma of the breast," (Cell 
61, 1121-1135). 
The 5-kbp EcoR1 k14-NT-3-hGH fragment (FIG. 1a) was isolated on 0.8% 
Seaplaque agarose gel (FMC Corporation), extracted from the gel using 
glassmilk purification (Geneclean Bio 101), and run through a NACS column 
(Bethesda Research Laboratories). 
The plasmid used to transfect mouse cells according to the present 
invention (K14-NT-3)-hGH has been deposited in E. coli with the American 
Type Culture Collection under the terms of the Budapest Treaty and is 
available as Accession No. 69889!. 
DNA was ethanol precipitated, resuspended in phosphate buffered saline at a 
concentration of 5 .mu.g/ml, and injected into fertilized B6.times.C3 F1 
hybrid mouse (Harlan Laboratory Supplies) embryos. Injections and 
implantations were carried out using standard procedures. (See Hogan, B., 
Costantini, F. & Lacy, E. (1986). Manipulating the Mouse Embryo: A 
Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). 
Mice were screened for the transgene using Southern hybridization analysis 
on DNA extracted from tail and by slot blot hybridization. For Southern 
hybridizations, 10 .mu.g of DNA was digested with BamH1, separated on an 
0.7% agarose gel, transferred to nitrocellulose by blotting and probed 
with a random primed .sup.32 P-dCTP (New England Nuclear) labeled probe 
made to the full length NT-3 cDNA. 
EXAMPLE 2 
Analysis of mRNA expression by Northern hybridization. Northern analysis 
was performed on RNA that was isolated from various tissues using 
guanidium thiocyanate/phenol/chloroform extraction. (See Chomczynski, P. & 
Sacchi, N. (1987), "Single-step method of RNA isolation by acid 
guanidinium thiocyanate-phenol-chloroform extraction," Analyt. Biochem. 
162, 156-159). 
Ten micrograms of total RNA was resolved on a 1.2% agarose formaldehyde 
denaturing gel, transferred to Nytran membrane (Schleicher and Schuell) by 
blotting, baked 2 h at 80.degree. C., and then hybridized to a .sup.32 
P-dCTP labeled riboprobe made to the full length NT-3 cDNA. 
EXAMPLE 3 
In situ hybridization. .sup.35 S-labelled cRNA probes were generated by 
adding 1 .mu.g of linearized to 2.5 mM each ATP, CTP, GTP, 15 mM .sup.35 
S!UTP (New England Nuclear) and either T7 or T3 polymerase (Stratagene) to 
generate antisense and sense probes, respectively. Incubation was for 60 
min at 40.degree. C. in transcription buffer containing 50 mM MgCl.sub.2, 
20 mM spermidine, 20 mM Tris (pH 7.4) and 10 mM DTT. The solution was 
phenol:chloroform extracted and precipitated with ammonium acetate and 2.5 
volumes of ethanol in the presence of 25 .mu.g carrier tRNA. 
For in situ hybridization, tail skin was dissected, immediately frozen on 
dry ice, cryostated at 20 .mu.m, mounted onto Glass plus slides and stored 
at -80.degree. C. until hybridized. To hybridize, slides were brought to 
room temperature, fixed for 10 min in 4% paraformaldehyde, washed in 
diethyl pyrocarbonate (DEPC)-treated phosphate buffer, transferred to 
0.25% acetic anhydride in 0.1M TEA (pH 8/0) for 10 min at room 
temperature, dehydrated through a graded series of alcohols and defatted 
in chloroform. Sections were hybridized for 12-24 hr at 60.degree. C. in a 
hybridization solution consisting of 1.times.10.sup.7 cpm/ml of .sup.35 
S-labeled cRNA probe, 50% formamide, 1.times. Denhardt's solution, 200 mM 
Tris (pH 7.5), 10% dextran sulfate, 0.3 .mu.g/ml salmon sperm DNA, 0.15 
mg/ml tRNA, and 40 mM DTT. Slides were washed in 4.times.SSC 
(1.times.SSC=0.15M NaCl, 0.015M sodium citrate, pH 7.0), incubated in 20 
mg/ml ribonuclease A (Sigma) dissolved in 10 mM Tris-saline, and then 
washed through descending concentrations of SSC. The final wash was with 
0.1.times.SSC at 37.degree. C. for 1 hour. 
Sections were air-dried and placed in X-ray cassettes with Hyperfilm 
.beta.-Max X-ray film (Amersham). Films were exposed for 3-7 days and 
developed in Kodak D-19. Selected slides were dipped in Kodak NTB-2 liquid 
emulsion, air-dried and exposed to film. Emulsion dipped slides were 
developed in D19, fixed in Kodak rapid fixer, counterstained with 
hematoxylin/eosin or cresyl violet and coverslipped in Permount. 
Skin sections from transgenic mice and non-transgenic siblings were 
processed in parallel. Controls for probe specificity included 
hybridization using a sense cRNA probe and pretreatment of tissue with 
RNase. Both of these conditions resulted in the absence of hybridization. 
EXAMPLE 4 
Immunohistochemistry. Skin from mice typically between 4-6 months of age 
was analyzed. Mice were deeply anesthetized with Avertin and 
transcardially perfused with 4% paraformaldehyde in phosphate buffer. 
Tissue was removed and immersion fixed for at least 4 h, placed in 25% 
sucrose overnight, embedded in gelatin and cut at 40 .mu.m thickness on a 
sliding microtome. Sections were blocked for 1 h in 5% normal goat serum 
(NGS), 2% bovine serum albumin (BSA), and 0.25% triton X-100 made in Tris 
buffered saline (100 mM Tris; 5 mM NaCl; pH 7.4) for 1 h, exposed 
overnight at room temperature to a primary antibody (anti-PGP 9.5, 1:5000; 
anti-NF150, 1:3000; dilutions made in 5% NGS and 0.25% triton), washed, 
and incubated in a 1:500 dilution of biotinylated goat anti-rabbit 
secondary for 1 h followed by a strep-avidin complex incubation (Vector 
Labs). Antibody binding was visualized using a nickel cobalt enhanced 
diaminobenzidine reaction. 
Merkel cell and touch dome counting. To identify touch domes and Merkel 
cells associated with touch domes located on flank skin of adult mice skin 
was depilated and 24 h later mice were injected intraperitoneally with 
quinacrine (Sigma) (15 mg/kg; made in phosphate buffered saline), a 
fluorescent dye that concentrates in neuroendocrine cell types (14). 12-20 
h following injection mice were deeply anesthetized and killed by cervical 
dislocation and flank skin removed. The skin was trimmed of connective 
tissue, mounted on a glass slike with a coverslip using glycerol, and 
Merkel cells associated with touch dome units counted using FITC 
fluorescence optics. The number of touch domes per cm.sup.2 of flank skin 
was determined in a similar manner. 
EXAMPLE 5 
NT-3 may be used in the diagnosis and/or treatment of neurologic disorders, 
including, but not limited to, peripheral neuropathies, such as diabetic 
neuropathies, toxic and nutritional neuropathies, hereditary neuropathies, 
and AIDS related neuropathies, as well as degenerative diseases such as 
amyotrophic lateral sclerosis (ALS). It has been shown that NT-3 supports 
the survival of dopaminergic neurons. Accordingly, in a preferred 
embodiment, NT-3 may be used for the diagnosis of Parkinson's disease. 
Because NT-3 has been observed to exhibit a spectrum of activity different 
from brain derived neurotrophic growth factor (BDNGF) and nerve growth 
factor (NGF), NT-3 provides new and valuable options for inducing the 
regrowth and repair of the central nervous system. 
The transgenic mice in accordance with the present invention, thus provide 
an in vivo model for testing drug interactions with neurons which are 
stimulated by NT-3. The K14-NT-3 mice are valuable to researchers that 
study the role of NT-3 in development and function of the adult nervous 
system. An area of intense study is the role of NT-3 in neurodegenerative 
disorders of the CNS and PNS. In addition, NT-3 has been shown to have 
antinociceptive effects (i.e., increase in tail-flick and hot plate 
response latency). Academic researchers as well as biotechnology companies 
are involved in such studies and would have interest in these mice as 
model systems in basic research studies as well as for applied studies 
such as drug testing. 
To test the reaction and/or of drugs in treating neurodegenerative 
disorders, a drug candidate is administered to a mouse according to the 
invention. The behavioral effects and tissue changes of the mouse are 
determined upon administration of the drug. 
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(1994). 
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12. M. S. Airaksinen, M. Koltzenburg, G. R. Lewin, submitted. 
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The purpose of the above description and examples is to illustrate some 
embodiments of the present invention without implying any limitation. It 
will be apparent to those of skill in the art that various modifications 
and variations may be made to the composition and method of the present 
invention without departing from the spirit or scope of the invention. All 
patents and publications cited herein are incorporated by reference in 
their entireties. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(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: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CCAGCGGGATCCGTGATGTCCATCTTGTTTTATGTG36 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CGGTACGGATCCGATGCCAATTCATGTTCTTCCG34 
__________________________________________________________________________