Materials and methods

Animals

Swiss random white (SRW) mice were purchased from the Animal Breeding Station, Mosgiel, NZ and used as embryo donors and recipients. At birth, a tail biopsy was taken from each mouse for DNA analysis.

Transgene constructs

Transgenic mice were made by pronuclear microinjection of a pKER-IGF-1 gene construct as previously described (^{Damak et al. 1996b}). The KER-IGF-1 DNA construct was made by ligating an ovine IGF-1 (oIGF-1) cDNA (^{Wong et al. 1989}) downstream from the mouse ultra-high sulfur keratin (UHS-KER) gene promoter (^{McNab et al. 1990}).

Screening for transgene integration

Mice born from microinjected embryos were tested for integration of the transgene by Southern blotting. Genomic DNA was extracted from tail biopsies taken shortly after birth according to the methods described by^{Sambrook et al. (1989)}. The extracted DNA was digested with appropriate restriction enzymes, separated on a 0.8% agarose gel electrophoresis and a Southern transfer performed as described by^{Sambrook et al. (1989)}. The oIGF-1 cDNA was random-prime labeled with ³²P using the Megaprime DNA labeling systems (Amersham, Bucks, U.K.) according to the manufacturer's instructions. Hybridization was performed with the oIGF-1 DNA probe labeled to a specific activity of 10⁸ dpm per g.

Analysis of transgene expression

Total RNA was extracted from mouse skin biopsies at 8 d (mid-anagen) and 15 d (late-anagen) of age using Trizol solution (Life Technologies, Gaithersburg, MD) according to the manufacturer's directions. Mice showing integration of the transgene were tested for expression of IGF-1 mRNA by ribonuclease protection assay (RPA) using a commercial kit (Ambion, Austin, TX). The DNA template used in RPA was prepared as previously described by^{Damak et al. (1996b)} The riboprobe contains 200 bp of the IGF-1 and 87 bp of vector sequences, which allowed the detection of a 287 bp band representing the transgenic message. RPA was accomplished by mixing 20 g total RNA with 10⁵ cpm ³²P-riboprobe according to the instructions from the Ambion kit.

Progeny testing

IGF-1 transgenic mice were produced by mating a transgenic SRW male expressing IGF-1 mRNA in the skin with a nontransgenic female. Food and water were provided ad libitum and fluorescent lighting from 5:00 am to 7:00 pm each day. At the birth of offspring, the sire was removed from the cage and the female kept in the same cage as the offspring throughout the trial. PCR was used to verify that offspring carried the IGF-1 transgene DNA in their genomes. DNA from mouse tails collected at 22 d of age was extracted as previously described and dissolved in TE buffer. This genomic DNA was mixed with 0.05 M of each primer KER (5'-TCCTGCTTATTGGTCCCTT-3') and IGF-1 (5'-AGGGCCAGATAGAAGAGATG-3') (synthesized by Oligos, Philadelphia, MA), 0.2 M each of dATP, dTTP, dCTP, and dGTP, 1 amplification buffer (10 mM Tris pH 8.3, 50 mM KCl, 2.5 mM MgCl₂) and 1 unit of Taq DNA polymerase (Promega, Madison, WI) in 50 l reactions. Amplification of DNA was achieved by 30 cycles of 94°C for 1 min, 55°C for 2 min, 72°C for 2.5 min, followed by 72°C for 7 min. Amplimers were analyzed using 1% agarose gel electrophoresis to determine whether the 350 bp UHS-KER-IGF-1 fragment was present. Total RNA extracted from skin biopsies of mice at day 22 was tested for expression of the transgene by RPA as previously described.

Measurement of vibrissa length

The incorporation of labeled cystine, as previously used to measure the rate of wool growth (^{Wynn et al. 1988}), was applied to the measurement of mouse vibrissa length. At 11, 16, and 21 d of age each mouse received a single, intraperitoneal injection of 0.25 Ci ³⁵S-L-cystine (specific activity 250 mCi per mM, Amersham) in 0.1 ml 0.9% (wt/vol) NaCl. Vibrissae were taken 24 h after the last injection, placed on microscope slides, coated with 5% (wt/vol) egg albumin, covered with photographic emulsion (Hypercoat Emulsions, Amersham), exposed at 4°C and developed 6 wk after exposure. Images of the fibers were projected at constant magnification through a photographic enlarger (Durst 300, Durst, Milan, Italy). The length between spots representing the first and second (days 11–16) and second and third (days 16–21) ³⁵S injections was measured with a planimeter to assess elongation during that period.

Statistical analysis

Inter-mouse variation was used to calculate the error of analysis of vibrissa elongation. Mean vibrissa length was calculated for each mouse at two different time periods (days 11–16 and 16–21) and the total period (days 11–21). The data were analyzed using the statistical program SYSTAT for Windows, Version 51992 (SYSTAT, Chicago, IL) running on a personal computer. Data arising from measurement of vibrissa length at days 11–16, 16–21, and 11–21 were subjected to analysis of covariance (ANCOVA) using body weight on day 22 as the covariate, to compare transgenic and nontransgenic mice. Data arising from measurements of body weight on day 22 were subjected to analysis of variance (ANOVA) to test effects of transgene expression.

Results

Microinjection and transfer of 435 embryos resulted in 40 mice being born, of which four were transgenic, 1377, 1417, 1453, and 1455, as determined by Southern blot analysis. Transgenesis was assessed by the presence of a 700 bp band that is not detectable in nontransgenic mouse DNA and each of the four transgenic mice had 5–10 unrearranged copies of the transgene (data not shown).

Gene expression

Transgene expression was found in skin at day 8 and day 15 of age in lines 1417, 1453, and 1455 as determined by RPA (data not shown). As the mice from line 1453 failed to produce offspring, further analysis for transgenic status and gene expression was performed from lines 1417 and 1455. Four of 12 mice from the same litter of line 1417 were transgenic as determined by PCR (Figure 1) and transgene expression was shown in the skin of three transgenic mice by RPA (Figure 2). As mice from line 1455 were all nonexpressors (data not shown), they were excluded from the analysis of vibrissa growth.

Figure 1.

Analysis of transgenic status by PCR in IGF-1 transgenic mice. Genomic DNA was extracted from mouse tails and amplified by PCR as outlined in Materials and Methods. Lane 1, a molecular weight size marker, lambda DNA digested with HindIII;lanes 2–13, DNA from 12 transgenic mice of line 1417 after PCR amplification and four (lanes 5, 6, 8, and 11) showed 350 bp UHS-KER-IGF-1 fragments;lane 14, DNA from one nontransgenic mouse of line 1377;lane 15, DNA from one nontransgenic mouse of line 1455;lane 16, DNA from one transgenic mouse of line 1455 and the 350 bp fragment is shown.

Full figure and legend (59K)

Figure 2.

Analysis of IGF-1 mRNA expression in the skin of mice from line 1417 by RPA. RPA was accomplished by hybridization of total RNA extracted from mouse skin with a radioactively labeled anti-sense IGF-1 riboprobe. The ovine IGF-1 probe protected a 287 bp fragment (lanes 6–8 and 10) corresponding to mRNA transcribed from the transgene (IGF-1 and vector) as described in Materials and Methods. Lane 1, partially degraded IGF-1 probe, which may be due to radiolytic decay;lane 2, a 330 nt mouse actin probe was prepared by in vitro transcription of a 250 bp mouse -actin DNA (provided with the RPA kit, Ambion) by T7 RNA polymerase according to the methods described in SP6/T7 Transcription Kit, Boehringer (Mannheim, Germany);lane 3, a 250 bp fragment corresponding actin message was protected by actin probe and detected in mouse liver;lane 4, some background is shown on the hybridization of IGF-1 probe with yeast RNA and this probably due to incomplete digestion of the probe by RNase;lane 5, blank lane;lanes 6–9, IGF-1 probe hybridized with transgenic mouse skin RNA from line 1417;lane 10, IGF-1 probe hybridized with mouse skin RNA from one expressor of line 1453.

Full figure and legend (59K)

Vibrissa elongation

Incorporation of ³⁵S-cystine into vibrissae from one of the transgenic mice from line 1417 is shown in Figure 3. Measurements were obtained on 10–15 vibrissae per mouse from both transgenic and nontransgenic groups. As there was only one mouse that was a nonexpressor in the transgenic group, analysis of vibrissa elongation for this mouse relative to those from other groups was not performed. Vibrissa elongation was compared between transgenic expressors and nontransgenics as shown in Table 1. Overexpression of IGF-1 in transgenic expressors had significant (p < 0.05) effect on vibrissa elongation at days 11–21. The increase in length is 16.6% compared with nontransgenics (Table 1). Vibrissa elongation in transgenics was also significantly (p < 0.05) increased compared with nontransgenics in two separate time periods: days 11–16 and days 16–21. The increase for transgenics over days 11–16 is 20.5% and days 16–21, 11.9% (Table 1).Table 1

Figure 3.

Measurement of vibrissa growth at days 11–16 and 16–21 of age in one of the transgenic line 1417 mice using ³⁵S-cystine incorporation into hair fibers. Three spots (from top) represent incorporation of ³⁵S-cystine into fibers of mouse vibrissae at days 11, 16, and 21 of age.

Full figure and legend (53K)

Body weight

Transgenic expressors did not differ (p > 0.05) from nontransgenics in body weight on day 22 of mouse age (Table 1).

Discussion

We previously reported increased fleece weights during yearling shearing in IGF-1 transgenic sheep compared with their nontransgenic half-sibs (^{Damak et al. 1996b}). In this paper, we report increased vibrissa length during the first neonatal hair cycle in transgenic mice compared with their littermates. The influence of IGF-1 on hair fiber growth in these transgenic animals may therefore be both increased fiber length and fiber weight. Further, our results demonstrated that, using ³⁵S-cystine, the length of vibrissa growth in mice can be determined by directly measuring the radioactively labeled spots being incorporated into the hair fibers (Figure 3).

The experiments were designed to increase local IGF-1 production in hair follicles by using a UHS-KER gene promoter. This promoter has been shown to direct a bacterial chloramphenicol acetyl transferase (CAT) gene in the skin and hair follicles of UHS-KER-CAT transgenic mice (^{McNab et al. 1990}). In addition, the CAT activity could only be detected in the anagen but not telogen phase of the hair cycle, suggesting that the UHS-KER gene contains the controlling elements for the correct tissue and developmental expression of the reporter CAT gene in transgenic mice (^{McNab et al. 1990}). The observation that dual mitogenic and morphogenic functions of IGF-1 in tissues (^{Sara & Hall 1990}) has led us to make UHS-KER-IGF-1 transgenic mice and attempt to enhance proliferation and differentiation of follicle cells by increased IGF-1 production.

Our results demonstrate that ectopic synthesis of IGF-1 in the skin may have local effects on vibrissa growth. This can be explained by the observation that elongated vibrissae were found at days 11–21 and consistent expression of the transgene from day 8 and days 15–22 of mouse age.^{Bol et al. (1997)} recently found that transgenic mice overexpressing IGF-1 in the epidermis have earlier hair follicle development at birth and this supports the local effect of IGF-1 on hair fiber growth. Although in situ hybridization was not performed in our studies to determine the precise localization of IGF-1 with reference to the follicle, expression of the transgene probably occurs in the keratogenous zone of follicle cells based on the detection of CAT expression in this zone in both UHS-KER-CAT transgenic mice and sheep (^{McNab et al. 1990;Damak et al. 1996a}).

The mechanism of action of the transgene may be due to the paracrine effects of IGF-1 on the proliferation of epithelial cells in the follicle bulb. IGF-1 is morphogenic and mitogenic to the skin and acts as a morphogen in the differentiation of hair follicles (^{Rudman et al. 1997}). This growth factor (^{Messenger 1989;Little et al. 1994b}), its receptors (IGF-1R) (^{Little et al. 1994a}) and its binding proteins (IGFBP) (^{Batch et al. 1996}) are expressed in the dermal papilla of hair follicles. In particular, the receptor was found in the epithelial matrix cells of follicles (^{Hodak et al. 1996}) and its protein is differentially expressed in the matrix cells through the hair cycle (^{Little et al. 1994a;Stones et al. 1994}). The papilla-derived IGF-1 may, therefore, have paracrine effects on the growth of epithelial matrix cells. In our study, expression of the transgene IGF-1 mRNA in the keratogenous zone may cause increased levels of IGF-1 which diffuses to the follicle bulb, changes local production of IGFBP, alters the interaction of transgene with IGF-1R and stimulates growth of epithelial cells in the follicle bulb, resulting in the observed phenotype. Although paracrine actions may be involved, it is also possible that overexpressed IGF-1 stimulates local follicular metabolism and enhances cysteine uptake (^{Harris et al. 1993}) which flows into increased vibrissa growth.

Systemic IGF-1 mediates the endocrine effects of GH and these effects may not extend to remote peripheral tissues such as skin or hair follicles. Using ³⁵S-cystine incorporation to hair fiber, we have found that intraperitoneal injection of IGF-1 into mice has no effect on vibrissa elongation in the anagen phase of the first hair cycle (Su, Damak, and Bullock, unpublished data). In wool growth, long-term systemic infusion of IGF-1 into sheep has been reported to have no effect on fiber growth rate (^{Cottam et al. 1992}) and injection of recombinant bovine or ovine GH into lambs results in no effect on wool growth despite elevated plasma IGF-1 (^{Spencer et al. 1994}). Further,^{Adams et al. (1996a)} demonstrate that the rate of wool growth is independent of circulating IGF-1 as its plasma levels are reduced while wool growth rates remain unaffected after animals were immunized against GH-releasing hormone during undernutrition. After a period of re-feeding, both control and immunized animals show a similar wool growth rate, although their plasma IGF-1 levels are different (^{Adams et al. 1996b}), suggesting that systemic IGF-1 is not an important determinant to wool growth. Recent findings from our IGF-1 transgenic sheep also support the less important role of systemic IGF-1 to wool growth. Plasma IGF-1 levels are similar between the UHS-KER-IGF-1 transgenic sheep and nontransgenic half-sibs while increased wool production was found in transgenics (^{Su et al. 1998}). Further studies in our UHS-KER-IGF-1 transgenic mice would require to measure the circulating level of IGF-1 and to determine its involvement on vibrissa growth.

It is surprising that vibrissa length is increased for transgenics at days 16–21 at the time when follicle growth proceeds to the catagen phase. We therefore speculate that overexpressed IGF-1 may have caused a delay in the onset of catagen, although confirmation would require histologic studies to determine the morphogenesis and differentiation of vibrissa follicles. In addition, a faster vibrissa growth rate at days 16–21 in both transgenic and control animals (Table 1), which may be due to the peripheral movement of labeled club hair over this period. It is thus important to undertake in vitro studies on the measurement of follicle growth to determine its relationship to fiber growth. Further, it would be important to compare the rates of DNA synthesis between transgenics and controls to determine their involvement in vibrissa fiber growth during days 11–21.

The IGF-1 transgenic mice failed to show enhanced body weight (Table 1), which may suggest that ectopic synthesis of IGF-1 in the skin did not produce a significant increase in the circulating IGF-1 of transgenic mice. The growth-promoting actions of GH are mediated by IGF-1 acting in both endocrine and paracrine/autocrine fashion, while GH has a dominant endocrine influence on plasma levels of IGF-1 and the majority of IGF-1 distributed in tissues is from plasma (^{Breier & Gluckman 1991}). Expression of the transgene in remote endocrine tissues such as skin and hair follicles may have less influence on plasma IGF-1 levels of transgenic mice, resulting in similar plasma levels of IGF-1 and, as a consequence, similar body growth in the two groups of animals.

In summary, the results presented here support the local effects of IGF-1 on vibrissa growth and keratinocyte-derived transgene IGF-1 may be involved in part of the effects through paracrine action to the epithelial matrix cells in the follicle bulb.

References

1. Adams, NR, Briegel, JR, Rigby, Rdg, Sanders, MR, Hoskinson, RM Responses of sheep to annual cycles in nutrition. 1. Role of endogenous growth hormone during undernutrition. Anim Sci, 1996a 62, 279–286,  | ISI | ChemPort |
2. Adams, NR, Sanders, MR, Briegel, JR, Peters, DW, Rigby, RdG Responses of sheep to annual cycles in nutrition. 2. Role of diet and endogenous growth hormone during replenishment. Anim Sci, 1996b 62, 287–292,  | ISI | ChemPort |
3. Barreca, A, deLuca, M, delMonte, P, et al. In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors. J Cell Physiol, 1992 151, 262–268,  | Article | PubMed | ISI | ChemPort |
4. Batch, JA, Mercuri, FA, Werther, GA Identification and localization of insulin-like growth factor-binding protein (IGFBP) messenger RNAs in human hair follicle dermal papilla. J Invest Dermatol, 1996 106, 471–475,  | Article | PubMed | ISI | ChemPort |
5. Behringer, RR, Lewin, TM, Quaife, CJ, Palmiter, RD, Brinster, RL, D'Ercole, AJ Expression of insulin-like growth factor I stimulates normal somatic growth in growth hormone-deficient transgenic mice. Endocrinology, 1990 127, 1033–1040,  | PubMed | ISI | ChemPort |
6. Bol, DK, Kiguchi, K, Gimenezconti, I, Rupp, T, Digiovanni, J Overexpression of insulin-like growth factor-1 induces hyperplasia, dermal abnormalities and spontaneous tumour formation in transgenic mice. Oncogene, 1997 14, 1725–1734,  | Article | PubMed | ISI | ChemPort |
7. Breier, BH & Gluckman, PD The regulation of postnatal growth: nutritional influences on endocrine pathways and function of the somatotrophic axis. Livest Prod Sci, 1991 27, 77–94,  | Article | ISI |
8. Cottam, YH, Blair, HT, Gallaher, BW, Purchas, RW, Breier, BH, McCutcheon, SN, Gluckman, PD Body growth, carcass composition, and endocrine changes in lambs chronically treated with recombinantly derived insulin-like growth factor-I. Endocrinology, 1992 130, 2924–2930,  | Article | PubMed | ISI | ChemPort |
9. D'Ercole, AJ, Stiles, AD, Underwood, LE Tissue concentrations of somatomedin C. Further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action. Proc Natl Acad Sci USA, 1984 81, 935–939,
10. Damak, S, Jay, NP, Barrell, GK, Bullock, DW Targeting gene expression to the wool follicle in transgenic sheep. Biotechnology, 1996a 14, 181–184,  | PubMed | ChemPort |
11. Damak, S, Su, H-Y, Jay, NP, Barrell, GK, Bullock, DW Improved wool production in transgenic sheep expressing insulin-like growth factor 1. Biotechnology, 1996b 14, 185–188,  | ChemPort |
12. Harris, PM, McBride, BW, Gurnsey, MP, Sinclair, BR, Lee, J Direct infusion of a variant of insulin-like growth factor-I into skin of sheep and effects on local blood flow, amino acid utilisation and cell replication. J Endocrinol, 1993 139, 463–472,  | PubMed | ISI | ChemPort |
13. Hodak, E, Gottlieb, AB, Anzilotti, M, Krueger, JG The insulin-like growth factor 1 receptor is expressed by epithelial cells with proliferative potential in human epidermis and skin appendages: Correlation of increased expression with epidermal hyperplasia. J Invest Dermatol, 1996 106, 564–570,  | Article | PubMed | ISI | ChemPort |
14. Ibrahim, L & Wright, EA The growth of rats and mice vibrissae under normal and some abnormal conditions. J Embryol Exp Morph, 1975 33, 831–844,  | PubMed | ISI | ChemPort |
15. Little, JC, Redwood, KR, Stones, AJ, Gibson, WT, Granger, SP The insulin-like growth factors is important in controlling the hair growth cycle. J Invest Dermatol, 1994a 102, 533 | ISI |
16. Little, JC, Westgate, GE, Evans, A Cytokine gene expression in intact rat hair follicles. J Invest Dermatol, 1994b 103, 715–720,  | Article | PubMed | ISI | ChemPort |
17. Mathews, LS, Hammer, RE, Behringer, RR, D'Ercole, AJ, Bell, GI, Brinster, RL, Palmiter, RD Growth enhancement of transgenic mice expressing human insulin-like growth factor-I. Endocrinology, 1988 123, 2827–2833,  | PubMed | ISI | ChemPort |
18. McNab, AR, Waldon, DJ, Buhl, AE, Vogeli, G Hair-specific expression of chloramphenicol acetyltransferase in transgenic mice under the control of an ultra-high-sulphur keratin promoter. Proc Natl Acad Sci USA, 1990 87, 6848–6852,  | PubMed | ChemPort |
19. Messenger, AG Isolation, culture and in vitro behaviour of cells isolated from the papilla of human hair follicles In: Van Neste, D Lachapelle, JM Antoine, JL, (eds). Trends in Human Growth, Alopecia Research. 1989 London: Kluwer Academic Publishers, pp. 57–67,
20. Philpott, MP, Sanders, DA, Kealey, T Effects of insulin-like growth factors on cultured human hair follicles: IGF-I at physiological concentrations is an important regulator of hair follicle growth in vitro. J Invest Dermatol, 1994 102, 857–861,  | Article | PubMed | ISI | ChemPort |
21. Robinson, M, Reynolds, AJ, Jahoda, CaB Hair cycle stage of the mouse vibrissa follicle determines subsequent fiber growth and follicle behavior In Vitro J Invest Dermatol, 1997 108, 495–500,  | ChemPort |
22. Rudman, SM, Philpott, MP, Thomas, GA, Kealey, T The roles of IGF-I in human skin and appendages – Morphogen as well as mitogen. J Invest Dermatol, 1997 109, 770–777,  | Article | PubMed | ISI | ChemPort |
23. Sambrook, J, Fritsch, EF, Maniatis, T Molecular Cloning – a Laboratory Manual. 1989 New York: Cold Spring, Harbor Laboratory Press
24. Sara, VR & Hall, K Insulin-like growth factors and their binding proteins. Physiol Rev, 1990 70, 591–614,  | PubMed | ISI | ChemPort |
25. Spencer, Gsg, Schurmann, A, Berry, C, Wolff, JE, Napier, JR, Hodgkinson, SC, Bass, JJ Comparison of the effects of recombinant ovine, bovine and porcine growth hormones on growth, efficiency and carcass characteristics in lambs. Livest Prod Sci, 1994 37, 311–321,  | Article | ISI |
26. Stones, AJ, Granger, SP, Jenkins, GLocalisation of cytokines and their receptors in human hair follicles using immunogold histochemistry. J Invest Dermatol 1994 102 (SID Abstract): 627,  | ISI |
27. Su, HY, Jay, NP, Gourley, TS, Kay, GW, Damak, S Wool production in transgenic sheep – results from first generation adults and second-generation lambs. Anim Biotechnol, 1998 9, 135–147,  | PubMed | ISI | ChemPort |
28. Sutton, R, Ward, WG, Raphael, KA, Cam, GR Growth factor expression in skin during wool follicle development. Comp Biochem Physiol, 1995 110B, 697–705,
29. Wong, EA, Ohlsen, SM, Godfredson, JA, Dean, DM, Wheaton, JE Cloning of ovine insulin-like growth factor-I cDNA. heterogeneity in the mRNA population. DNA, 1989 8, 649–657,  | PubMed | ISI | ChemPort |
30. Wynn, PC, Wallace, Alc, Kirby, AC, Annison, EF Effects of growth hormone administration on wool growth in Merino sheep. Aust J Biol Sci, 1988 41, 177–187,  | PubMed | ISI | ChemPort |

Acknowledgments

We thank Barry Palmer for his comments on the manuscript. The work was supported in part by the University Research Trust of Lincoln University.