Exp Anim. 2017 Jul 5 

Transgenic mice display hair loss and regrowth overexpressing mutant Hr gene

Cunshuan Xu


摘要:

Mutations in the hairless (Hr) gene in both mice and humans have been implicated in the development of congenital atrichia, but the role of Hr in skin and hair follicle (HF) biology remains unknown. Here, we established transgenic mice (TG) overexpressing mutant Hr to investigate its specific role in the development of HF. Three transgenic lines were successfully constructed, and two of them (TG3 and TG8) displayed a pattern of hair loss and regrowth with alternation in the expression of HR protein. The mutant Hr gene inhibited the expression of the endogenous gene in transgenic individuals, which led to the development of alopecia. Interestingly, the hair regrew with the increase in the endogenous expression levels resulting from decreased mutant Hr expression. The findings of our study indicate that the changes in the expression of Hr result in hair loss or regrowth.Transgenic founder mice were produced on a C57BL/6 background. Full-length cDNAs of the hairless gene (Hr) were cloned, and a point mutation (G3427A, RefSeq NM_021877) was introduced as in our previous study [12]. The entry clones (pDown-mHairless) were obtained by BP reaction cloning (Invitrogen) between attB1-mHairless/IRES/eGFP-attB2 and the entry vector of pDonr221 according to the instructions for Gateway® BP Clonase™ II Enzyme Mix (Invitrogen). The expression vector pRP (Exp)-EF1A>mHairless mutant>IRES/EGFP was successfully constructed by Gateway LR reactions according to manufacturer’s recommendation for Gateway® LR Clonase™ II Plus Enzyme Mix (Invitrogen) between the entry clones and pRP.Des3d vector (TGBS140526Q1, Cyagen Biosciences Inc., Guangzhou, China). The recombinant vector synthesized by linearization with NotI was purified from agarose gel using a QIAquick Gel Extraction Kit (Qiagen, Chatsworth, CA, USA) and adjusted to a final concentration of 3 ng/µl in 10 mM Tris/0.1 mM EDTA (pH 7.4) buffer for microinjection. Male C57BL/6 mice were mated with female C57BL/6 mice that were superovulated with exogenous gonadotrophins, and 196 fertilized eggs were collected from the oviducts the next morning. Transgenic mice were obtained by microinjection of the linearized DNA solution into fertilized oocytes and were identified by PCR analysis with Hr-specific primers (mHairless-F, 5′-CTTGGCACTTGATGTAAT TCTCC-3′, and mHairless-R, 5′-CCCACAATGCCGTTCACAGG-3′; PCR fragment=220bp) under the following conditions: 30 cycles of 94°C for 30 s, 58°C for 35 s and 72°C for 35 s, and then a final extension step of 72°C for 5 min.Using PCR analysis, seven founders were found to contain the mutant Hr gene in their chromosomal DNA. Then, transgenic lines were established by mating with the C57BL/6 strain. Next, 5 of F1 animals shown to be transgenic for mutant Hr were intercrossed to produce F2. Five-eight F3 mice were then generated by the same method. Theoretically, a transgenic mouse homozygous for mutant Hr can be confirmed if all the offspring are found to be mutant Hr positive through mating of the mouse to wild-type partners according to Mendelian principles.All procedures performed in studies involving animals were in accordance with the ethical standards approved by the Animal Ethics Committee of Zhengzhou University. Animals were maintained on a 12-h light: 12-h dark circadian schedule in a 20–24°C housing room and were provided water and mouse chow ad libitum.Transgenic mice were obtained by microinjection of the following constructs into fertilized oocytes (Fig. 1A). Of 26 pups recovered, seven (referred to as mice #3, #5, #7, #8, #12, #16, and #21) were found to contain the mutant Hr gene in their chromosomal DNA using PCR analysis (Fig. 1B). Seven independent lines were subsequently established by mating with C57BL/6 strains, but only three lines (TG3, TG8, and TG12) were successfully constructed. The other lines could not be established due to death or infertility. Approximately 79% F3 mice (46/58) had an abnormal phenotype with hair loss in the T3 and T8 lines, while the hair developed normally in all the F1 and F2 pups. By mating the mice with hair loss with C57BL/6 strains, all of their offspring were shown to be transgenic for mutant Hr. Forty-six F3 mice with hair loss were confirmed to be homozygous for mutant Hr(see the Materials and Methods section for the judgment method) and used in the study.We appreciate the support of the State Key Laboratory Breeding Base Co-sponsored by Henan & the Ministry of Science and Technology for Cell Differentiation Regulation for this work. This work was supported by the grants from the National Natural Science Foundation of China (No. 39970119 and 31372270).
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