Keratin-14 Promoter Research Articles

Differential Dependency of Unmyelinated and Aδ Epidermal and Upper Dermal Innervation on Neurotrophins, Trk Receptors, and p75LNGFR

The impact of the nerve growth factor (NGF) family of neurotrophins and their receptors was examined on the cutaneous innervation in the mystacial pads of mice. Ten sets of unmyelinated and thinly myelinated sensory and autonomic innervation were evaluated that terminated in the epidermis, upper dermis, and upper part of the intervibrissal hair follicles. Mystacial pads were analyzed from newborn to 4-week-old mice that had homozygous functional deletions of the genes for NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), tyrosine kinase (trk) A, trkB, trkC, or p75. Mystacial pads were also analyzed in adult transgenic mice that had overproduction of NGF, BDNF, or NT-3 driven by a keratin promoter gene. The innervation was revealed by using immunofluorescence and immunocytochemistry with antibodies for protein gene product (PGP) 9.5, calcitonin gene-related product (CGRP), substance P (SP), galanin (GAL), neuropeptide Y (NPY), tyrosine hydroxylase (TH), and a neurofilament protein. The cumulative results indicated that NGF/trkA signaling plays a major role in the outgrowth and proliferation of sensory axons, whereas NT-3/trkA signaling plays a major role in the formation of sensory endings. TrkC is also essential for the development of three sets of trkA-dependent sensory innervation that coexpress CGRP, SP, and GAL. Another set of sensory innervation that only coexpressed CGRP and SP was solely dependent upon NGF and trkA. Surprisingly, most sets of trkA-dependent sensory innervation are suppressed by trkB perhaps interacting with p75. BDNF and NT-4 appear to mediate this suppressing effect in the upper dermis and NT-4 in the epidermis. In contrast to sensory innervation, sympathetic innervation to the necks of intervibrissal hair follicles depends upon NGF/trkA signaling interacting with p75 for both the axon outgrowth and ending formation. Although NT-3/trkA signaling is essential for the full complement of sympathetic neurons, NT-3 is detrimental to the formation of sympathetic terminations to the necks of hair follicles. TrkB signaling mediated by BDNF but not NT-4 also suppresses these sympathetic terminations. One sparse set of innervation, perhaps parasympathetic, terminating at the necks of hair follicles is dependent solely upon NT-3 and trkC. Taken together, our results indicate that the innervation of the epidermis, upper dermis, and the upper portion of hair follicles is regulated by a competitive balance between promoting and suppressing effects of the various neurotrophins.

The production of a transgenic rat expressing nerve growth factor using cell-type specific keratin promoters.

The production of a transgenic rat expressing nerve growth factor using cell-type specific keratin promoters.

Decreased skin sensory innervation in transgenic mice overexpressing insulin-like growth factor-II

Cutaneous sensory innervation was studied in transgenic mice overexpressing insulin-like growth factor II using a keratin promoter. The skin area of these animals is enlarged [54]providing increased target for sensory neurons. L4 dorsal root ganglion cell counts revealed that the total number of sensory neurons was the same in transgenics as control animals. Levels of nerve growth factor per unit weight of skin were also unchanged. The cutaneous nerves of the hindlimb were immunostained with the pan-neuronal marker PGP 9.5 in transgenic and control mice at postnatal day 0 and 21. The innervation in transgenic mice was markedly reduced, particularly in superficial dermis and epidermis and in some areas innervation was completely absent. The effect was greatest in distal skin regions and increased with age. Since insulin-like growth factor II has been reported to be a sensory neurotrophic factor, [42]its effect on neurite outgrowth was tested on embryonic day 14 and 18 mouse lumbar dorsal root ganglion explants in culture. Under these conditions insulin-like growth factor II (5–100 ng/ml) did not have strong growth promoting activity and at embryonic day 18, in the presence of 5–10 ng/ml nerve growth factor, neurite outgrowth was suppressed by insulin-like growth factor II. The results show that increased skin target and availability of nerve growth factor per se do not alter the number of innervating sensory neurons. However, reduced sensory terminal arborization and skin hypoinnervation does occur in the presence of excess insulin-like growth factor-II. It is possible that insulin-like growth factor-II inhibits terminal axon growth directly via receptors on sensory neurons or peripheral glia.

Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 transgene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and impairs keratinocyte proliferation.

CD44 is a broadly distributed polymorphic glycoprotein that serves as the principal cell-surface receptor for hyaluronate. Although CD44-mediated cell interaction with hyaluronate has been implicated in a variety of physiologic events, including cell-cell and cell-substrate adhesion, cell migration, proliferation, and activation, as well as hyaluronate uptake and degradation, the biologic role of CD44 in vivo in various tissues remains to be determined. In the present work we have developed transgenic mice that express an antisense CD44 cDNA driven by the keratin-5 promoter. These mice lack detectable CD44 expression in skin keratinocytes and corneal epithelium and display abnormal hyaluronate accumulation in the superficial dermis and corneal stroma, distinct morphologic alterations of basal keratinocytes and cornea, and defective keratinocyte proliferation in response to mitogen and growth factors. These alterations are reflected by a decrease in skin elasticity, impaired local inflammatory response and tissue repair, delayed hair regrowth, and failure of the epidermis to undergo hyperplasia in response to carcinogen. Our observations indicate that two major functions of CD44 in skin are the regulation of keratinocyte proliferation in response to extracellular stimuli and the maintenance of local hyaluronate homeostasis.

In vivo footprints are found in the Xenopus 63 kDa keratin gene promoter prior to the appearance of mRNA

Previous work on the promoter region of the 63 kDa keratin gene demonstrated that in vivo footprints did not change during the transition from low-level to high-level transcription. Reverse transcription polymerase chain reaction and in vivo footprinting were used to determine whether these DNA–protein interactions are present before transcription begins. The results presented indicate that during development, DNA–protein interactions are present in the promoter region of the 63 kDa keratin gene at stage 44, four days prior to the initial appearance of 63 kDa keratin mRNA, at stage 48. Thus, the occupancy of these sites at stage 44 is not sufficient for transcription, but may have a role in `poising' the keratin promoter for the initiation of epidermal-specific transcription. The results suggest that the developmental history of a gene may be important in regulating its temporal and spatial expression.

Mouse Vaginal Opening Is an Apoptosis-Dependent Process Which Can Be Prevented by the Overexpression of Bcl2

In the mouse, opening of the vaginal cavity to the skin is a late event, occurring around the fifth week of life; it can be induced in sexually immature mice by β-estradiol injections. We have generated two lines of transgenic mice expressing the human Bcl2 protein in a variety of tissues. The vaginal cavity of the transgenic females remained permanently closed, a condition completely resistant to β-estradiol injections; this was accompanied by a considerable distension of the genital tract. Histologic studies of vaginal sections at the time of opening to the skin in normal mice showed, by the TUNEL method which detects nuclei with fragmented DNA characteristic of apoptosis, that this event coincides with extensive apoptosis in the lower part of the vaginal mucosa, a process prevented in the bcl2 transgenic mice, which express Bcl2 in suprabasal epithelial cells and in subepithelial cells of the vaginal mucosa. In contrast, two lines of mice bearing a Bcl2 transgene placed under the control of a K10 keratin promoter, whose expression is restricted to the suprabasal layers of the epidermis, had a normal phenotype. Eyelids’ formation and opening of the external ear canals, which also occur after birth in the mouse, were not altered in any of these transgenic lines; histological study of eye and ear sections at the time of these events failed to detect apoptosis. In conclusion, the tissue remodeling required to complete maturation of the mouse female genital tract at the time of puberty is an hormonally triggered apoptosis-dependent process.

Effects of NGF overexpression on anatomical and physiological properties of sympathetic postganglionic neurons

To examine the effects of increased target derived nerve growth factor (NGF) on the sympathetic nervous system, the superior cervical ganglion was characterized in transgenic mice overexpressing NGF in keratinized epithelium (e.g. skin, tongue and oral cavity). In these mice NGF overexpression was achieved via expression of an NGF transgene driven by the K14 keratin promoter. This promoter is expressed at approximately embryonic day 11 and thereafter expressed constitutively in the adult. This expression results in supranormal levels of NGF in targets of sympathetic postganglionic neurons prior to the period of programmed cell death. Examination of postnatal day 6 (PN6) and adult transgenic mice shows ca. 2.5-fold increase in neuron number in the superior cervical ganglion (SCG). Analysis of SCG neuronal size revealed a dramatic hypertrophy in the transgenic mice that is present by PN6 and is maintained in the adult. Intracellular physiological measurements of control superior cervical ganglia identified two distinct types of neurons identified on the basis of their response to depolarizing current; ‘phasic’ neurons fire a single action potential while ‘tonic’ neurons fire continuously. In adult transgenic mice the phasic; neurons were 102% larger than control phasic neurons while the tonic neurons only increased 44% relative to controls. The hypertrophy of sympathetic ganglia in the transgenic mice was correlated with an increased innervation of skin and dorsal root ganglia, structures that either express the transgene or concentrate NGF produced by the skin.

Chronic estrogen-induced cervical and vaginal squamous carcinogenesis in human papillomavirus type 16 transgenic mice.

High-risk human papillomaviruses (HPVs), including type 16, have been identified as factors in cervical carcinogenesis. However, the presence and expression of the virus per se appear to be insufficient for carcinogenesis. Rather, cofactors most likely are necessary in addition to viral gene expression to initiate neoplasia. One candidate cofactor is prolonged exposure to sex hormones. To examine the possible effects of estrogen on HPV-associated neoplasia, we treated transgenic mice expressing the oncogenes of HPV16 under control of the human keratin-14 promoter (K14-HPV16 transgenic mice) and nontransgenic control mice with slow release pellets of 17beta-estradiol. Squamous carcinomas developed in a multistage pathway exclusively in the vagina and cervix of K14-HPV16 transgenic mice. Estrogen-induced carcinogenesis was accompanied by an incremental increase in the incidence and distribution of proliferating cells solely within the cervical and vaginal squamous epithelium of K14-HPV16 mice. Expression of the HPV transgenes in untreated transgenic mice was detectable only during estrus; estrogen treatment resulted in transgene expression that was persistent but not further upregulated, remaining at low levels at all stages of carcinogenesis. The data demonstrate a novel mechanism of synergistic cooperation between chronic estrogen exposure and the oncogenes of HPV16 that coordinates squamous carcinogenesis in the female reproductive tract of K14-HPV16 transgenic mice.

Targeting gene expression to the wool follicle in transgenic sheep.

To establish the feasibility of overexpressing foreign genes in the wool follicle, transgenic sheep were produced by pronuclear microinjection of a DNA construct consisting of a mouse ultrahigh-sulfur keratin promoter linked to the bacterial chloramphenicol acetyl transferase (CAT) gene. Four of 31 lambs born were transgenic. The overall efficiency of transgenesis was 1.1% of zygotes injected and transferred. Two transgenic rams were mated to nontransgenic ewes, and both transmitted the gene to their offspring in Mendelian fashion. CAT expression was found in the skin of one G0 ram and in 9 out of 26 transgenic G1 progeny. Two G1 lambs were sacrificed to study tissue specificity. Both had high levels of expression in skin but One had high expression in spleen and kidney with lower levels of expression in lung; the other had low expression in spleen, lung, and muscle. In situ hybridization demonstrated that transgene expression in the skin was confined to the keratogenous zone of the wool follicle cortex. Expression of CAT activity in skin was correlated with diet-induced or seasonal changes in the rate of wool growth. This keratin promoter appears useful for overexpressing factors in the wool follicle that might influence wool production or properties.

Improved wool production in transgenic sheep expressing insulin-like growth factor 1.

Transgenic sheep were produced by pronuclear microinjection with a mouse ultra-high-sulfur keratin promoter linked to an ovine insulin-like growth factor 1 (IGF1) cDNA. Five transgenic lambs resulted from the microinjection of 591 embryos; one male and one female showed IGF1 expression in the skin. A progeny test of the ram was carried out by matings to 43 non-transgenic ewes. Of 85 lambs born, 43 (50.6%) were transgenic. At yearling shearing (approximately 14 months of age), clean fleece weight was on average 6.2% greater in transgenic animals than in their non-transgenic half-sibs, with a greater effect in males (9.2%) than females (3.4%). Transgenics showed a small but significant increase in bulk, but male transgenics had a lower staple strength than female transgenics and non-transgenics which did not differ significantly. There were no significant differences in fiber diameter, medullation, and hogget body weight. To our knowledge this is the first reported improvement in a production trait by genetic engineering of a farm animal without adverse effects on health or reproduction.

Overexpression of anAgouticDNA in the Skin of Transgenic Mice Recapitulates Dominant Coat Color Phenotypes of Spontaneous Mutants

The classical mouse fancyAgoutigene is responsible for the wild-type coat color where hairs are banded black and yellow. TheAgoutigene encodes a 131-amino-acid secreted protein product that regulates phaeomelanin synthesis by melanocytes in mice. Mice with a dominant mutation at this locus,Ay, develop a yellow coat color, obesity, and diabetes, as the result of a deletion that results in ectopic overexpression of theAgoutigene mRNA in all tissues examined. Obesity and diabetes inAymutant mice could be caused by circulation of the protein, or localized action in specific tissues as a paracrine factor acting in cell–cell communication. To test these two possibilities, theAgouticDNA was overexpressed in the skin of transgenic mice using either theTyrosinase-Related Protein-1or the keratin-14 (K14) promoter, the latter with and without an intron. The K14 promoter directed high constitutive levels of expression ofAgoutimRNA in the skin, and several lines of transgenic mice exhibited coat colors resembling dominantAgoutiallele phenotypes. Two highly expressing K14–Agouti transgenic lines, with light-yellow pelage, were analyzed for obesity and hyperglycemia. The transgenic mice were not significantly different from the controls (P> 0.05), indicating that theAgoutiproduct does not act as an endocrine factor. RNase protection assays revealed a correlation between the levels of dorsal and ventral skin expression with pigmentation/phaeomelanin phenotypes. Co-injection experiments with the Agouti transgenes and other transgenes demonstrated co-integration of the two constructs at the same chromosomal site in approximately 95% of F1 progeny, allowing transgene inheritance to be visibly detected.

Stomach cancer in transgenic mice expressing human papillomavirus type 16 early region genes from a keratin promoter

Microbiology Society journals contain high-quality research papers and topical review articles. We are a not-for-profit publisher and we support and invest in the microbiology community, to the benefit of everyone. This supports our principal goal to develop, expand and strengthen the networks available to our members so that they can generate new knowledge about microbes and ensure that it is shared with other communities.

Age-dependent phenotypic switching of mast cells in NGF-transgenic mice.

Effects of overexpression of nerve growth factor (NGF) on mast cell phenotype and numbers were investigated in nasal and oral mucosae and skin of 3- and 6-week-old transgenic mice in which NGF expression in epithelial basal cells was driven by the keratin-14 promoter. Mast cell phenotypes were identified by Alcian blue/safranin and berberine sulfate histochemistry. In the 3-week-old transgenic mice, NGF overexpression had no effect on phenotype except in tongue, where mast cells exhibited mixed or connective tissue phenotypes compared with the mucosal phenotype in the non-transgenic. In 6-week-old transgenic animals, NGF overexpression resulted in the mucosal phenotype in tissues which contained connective tissue or mixed mast cells in non-transgenics. Mast cell hyperplasia occurred at both ages. NGF effects on mast cell phenotype were age-dependent and involve complex microenvironmental interactions.

Circulating human factor IX produced in keratin-promoter transgenic mice: a feasibility study for gene therapy of haemophilia B.

It has previously been suggested that keratinocytes might provide a suitable target cell for delivery of factor IX to the systemic circulation for gene therapy of haemophilia B. Here, an investigation of the use of cellular gene promoters specific for keratinocytes was undertaken to examine whether factor IX could be passed from the epidermis to the systemic circulation. Utilizing two bovine cytokeratin gene promoters, BKIII and BKVI, three lines of transgenic mice were generated with targeted expression of human factor IX in the epidermis. All three transgenic mouse lines secreted epidermally derived human factor IX into the blood system. Most effective factor IX expression (46 ng/ml steady-state levels of circulating human factor IX) was obtained utilizing the BKVI gene promoter, the human homologue of K10, which is expressed exclusively in differentiated keratinocytes, localized distal to the basement membrane. This report demonstrates, for the first time, that human factor IX can be efficiently synthesized and secreted from keratinocytes in situ, and can cross the epidermal basement membrane to reach the systemic circulation. The transgenic mouse model will provide a good in vivo system with which to optimize the efficiency of different keratin gene promoter constructs for delivery of therapeutic gene products to the serum, especially for those promoters, such as K10, which are not effectively expressed in vitro.

Epidermal expression of intercellular adhesion molecule 1 is not a primary inducer of cutaneous inflammation in transgenic mice.

Keratinocytes at sites of cutaneous inflammation have increased expression of intercellular adhesion molecule 1 (ICAM-1), a cytokine-inducible adhesion molecule which binds the leukocyte integrins LFA-1 and Mac-1. Transgenic mice were prepared in which the expression of mouse ICAM-1 was targeted to basal keratinocytes by using the human K14 keratin promoter. The level of constitutive expression attained in the transgenic mice exceeded the peak level of ICAM-1 expression induced on nontransgenic mouse keratinocytes in vitro by optimal combinations of interferon gamma and tumor necrosis factor alpha or in vivo by proinflammatory stimuli such as phorbol 12-myristate 13-acetate. In vitro adhesion assays demonstrated that cultured transgenic keratinocytes were superior to normal keratinocytes as a substrate for the LFA-1-dependent binding of mouse T cells, confirming that the transgene-encoded ICAM-1 was expressed in a functional form. However, the high level of constitutive ICAM-1 expression achieved on keratinocytes in vivo in these transgenic mice did not result in additional recruitment of CD45+ leukocytes into transgenic epidermis, nor did it elicit dermal inflammation. Keratinocyte ICAM-1 expression also did not potentiate contact-hypersensitivity reactions to epicutaneous application of haptens. The absence of a spontaneous phenotype in these transgenic mice was not the result of increased levels of soluble ICAM-1, since serum levels of soluble ICAM-1 were equal in transgenic mice and controls. We conclude that elevated ICAM-1 expression on keratinocytes cannot act independently to influence leukocyte trafficking and elicit cutaneous inflammation.

Altered expression of nerve growth factor in the skin of transgenic mice leads to changes in response to mechanical stimuli

It has recently become clear that the neurotrophic factor, nerve growth factor, interacts specifically with nociceptive sensory neurons during development and maturity. Indeed, it may serve as a critical link between inflammation and the hyperalgesia that ensues in adult animals. Nerve growth factor is normally expressed in limiting amounts in target tissues of sensory and postganglionic sympathetic neurons. In the present study we have altered the basal level of nerve growth factor expression in the skin by producing transgenic mice that express a fusion gene construct containing either a sense or antisense nerve growth factor complementary DNA linked to the K14 keratin promoter. The K14-nerve growth factor transgene (sense or antisense) is abundantly expressed in skin from approximately embryonic day 15 and is then constitutively expressed throughout the life of the animal. In light of the fact that systemic administration of nerve growth factor to neonatal or adult rats leads to hyperalgesia, we have asked whether mice expressing the sense K14-nerve growth factor transgene exhibit similar sensory abnormalities and whether mice expressing the antisense nerve growth factor complementary DNA were hypoalgesic. Here we show that mice over-expressing nerve growth factor in skin display a profound hyperalgesia to noxious mechanical stimulation. Additionally, K14-nerve growth factor antisense mice displayed a profound hypoalgesia to the same stimuli.

Epidermal growth factor and transforming growth factor alpha specifically induce the activation- and hyperproliferation-associated keratins 6 and 16.

Epidermal injury results in activation of keratinocytes which produce and respond to growth factors and cytokines and become migratory. Activated keratinocytes express a specific pair of keratin proteins, K6 and K16, distinct from the keratins in the healthy epidermis. Keratinocytes can be activated, for example, by binding of the appropriate ligands to the epidermal growth factor receptor (EGFR). We have analyzed the effects of EGFR activation on keratin gene transcription by transfecting DNAs containing keratin promoters linked to a reporter gene into primary cultures of human epidermal keratinocytes in the presence or absence of EGF or transforming growth factor alpha (TGF alpha), two growth factors that activate EGFR. The activation of EGFR had no effect on the promoters of simple epithelial, basal-layer-specific, or differentiation-specific keratins. In contrast, the expression of K6 and K16 was strongly and specifically induced. A 20-bp DNA segment of the K16 gene promoter conveyed the EGF regulation, functioned in a heterologous construct, and therefore constituted an EGF-responsive element. A nuclear protein specifically bound to this element and to the analogous sequence of the K6 promoter. Thus, EGF specifically induces K6 and K16, markers of activated keratinocytes, via nuclear proteins that bind to EGF-responsive elements in the promoters of these keratin genes.

Cachexia and graft-vs.-host-disease-type skin changes in keratin promoter-driven TNF alpha transgenic mice.

Tumor necrosis factor alpha (TNF alpha) orchestrates a wide range of effects that combat severe infections in animals. At lower levels, TNF alpha plays an important protective role in stimulating chemotaxis and antimicrobial activity of neutrophils, macrophages, and eosinophils. During chronic illness, TNF alpha secretion can be elevated markedly, giving rise to cachexia, hemorrhage, necrosis and, ultimately, death. Although TNF alpha may mediate many of its effects through macrophages, 30% of TNF alpha injected into animals concentrates in the skin. In recent years, it has been shown that keratinocytes can be induced to synthesize TNF alpha. To explore the role of TNF alpha synthesis in keratinocytes, we used a keratin-14 (K14) promoter to target human TNF alpha expression in the epidermis and other stratified squamous epithelia of transgenic mice. Most mice expressing the K14-TNF alpha transgene stopped gaining weight within 1 week postbirth, and exhibited retarded hair growth. In the skin, adipose production was profoundly inhibited, whereas signs of fibrosis and immune infiltration were evident in the dermis. Over time, the epidermis exhibited an increased stratum corneum, as signs of necrosis began to appear in the skin. Within 3-5 weeks, the mice displayed features characteristic of cachexia and necrosis. Our results suggest that TNF alpha expression by keratinocytes not only plays a role in inflammatory and graft-versus-host-disease-like responses in the skin, but also in other tissues, apparently by virtue of stratified squamous epithelial-derived TNF alpha entering the bloodstream. Our results have enabled the first evaluation of many of the effects of TNF alpha in transgenic animals.

Regulation of Keratin Gene Expression: The Role of the Nuclear Receptors for Retinoic Acid, Thyroid Hormone, and Vitamin D3

Keratinization, the orderly process of differentiation of epidermal keratinocytes from stratum basale to stratum corneum, is influenced by hormones and vitamins. We have used expression of epidermal keratins as a paradigm of keratinization processes and analyzed the effects of retinoic acid, thyroid hormone, and vitamin D3 on keratin gene expression. DNA constructs in which keratin gene promoters drive expression of reporter genes were co-transfected with vectors expressing nuclear receptors for the above molecules into various cell types. The keratin promoters studied included K3, K5, K10, K14, and K16. The recipient cell types were HeLa and primary cultures of rabbit corneal and esophageal epithelial cells and of human epidermal keratinocytes. We found that retinoic acid, via its nuclear receptor, suppresses expression of all the above-listed keratin genes. Thyroid hormone and its receptor similarly suppressed those genes. The site of interaction between these two receptors and the promoter sequences of K10 and K14 genes has been identified. Surprisingly, vitamin D3 and its receptor had no direct effect on keratin promoters. Our results suggest that a retinoic acid has a twofold effect on keratin gene expression: by regulating keratinocyte differentiation it determines which keratins are expressed, basal cell specific or differentiation specific; by direct interaction between its receptor and keratin genes, retinoic acid determines the total amount of keratin protein within the cell. Vitamin D3, on the other hand, also regulates keratinocyte differentiation, but does not directly interact with the keratin genes.

MYC Protooncogenes of Wool and Hair Growtha

The growth of hard keratin fibers such as wool and hair is dependent on the proliferation of cells in the follicle bulb. If the cells leaving the bulb could be induced to undergo an extra division, then fiber growth should increase. The cellular division within the follicle is complex and probably involves one or more growth factors, which act by altering the expression of transcription factors and other nuclear proteins. We propose that the expression of the myc protooncogenes is a central part of this mechanism. In support of this hypothesis we have detected the mRNAs for TGF-beta 1, basic FGF, TGF-alpha, and c-myc in plucked wool follicles using PCR amplification. We have also shown that the TGF-beta 1, TGF-beta 2, TGF-beta 3, EGF, TGF-alpha, basic FGF, N-myc, and c-myc genes are expressed in mouse skin, and we looked for changes during the hair cycle. The PCR data suggest that in whole skin the levels of mRNA for TGF-beta 1, TGF-beta 2, TGF-alpha, and c-myc do not change. In Quackenbush mice the levels for N-myc, TGF-beta 3, and basic FGF mRNA appear to be lower at the end of the hair cycle. We have confirmed in CBA/C57 black mice that lower levels of N-myc mRNA are detected when hair growth ceases in catagen and telogen. To test our hypothesis further and to assess its practical application, we are making transgenic mice in which the N-myc gene is overexpressed in the hair follicle by way of a wool keratin promoter. The transgene consists of 3.3 kb of 5' sequence from an ovine type 1 IF gene, the murine N-myc genomic coding sequence, and an SV40 polyadenylation signal. The native keratin type 1 IF gene is expressed exclusively in the wool follicle, as shown by in situ hybridization. However, in mice the injection of the transgene has resulted in high embryonic mortality and some embryos with large body size and head malformations. Since these mice were not transgenic, this is likely to be an effect of transient expression of the transgene during embryogenesis. The two transgenic mice produced so far have a normal phenotype.