Hairless-knockout piglets generated using the clustered regularly interspaced short palindromic repeat/CRISPR-associated-9 exhibit abnormalities in the skin and thymus

Abstract

The nuclear receptor corepressor Hairless (HR) interacts with nuclear receptors and controls expression of specific target genes involved in hair morphogenesis and hair follicle cycling. Patients with HR gene mutations exhibit atrichia, and in rare cases, immunodeficiency. Pigs with HR gene mutations may provide a useful model for developing therapeutic strategies because pigs are highly similar to humans in terms of anatomy, genetics, and physiology. The present study aimed to knockout the HR gene in pigs using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated-9 (Cas9) system and to investigate the molecular and structural alterations in the skin and thymus. We introduced a biallelic mutation into the HR gene in porcine fetal fibroblasts and generated nine piglets via somatic cell nuclear transfer. These piglets exhibited a lack of hair on the eyelids, abnormalities in the thymus and peripheral blood, and altered expression of several signaling factors regulated by HR. Our results indicate that introduction of the biallelic mutation successfully knocked out the HR gene, resulting in several molecular and structural changes in the skin and thymus. These pigs will provide a useful model for studying human hair disorders associated with HR gene mutations and the underlying molecular mechanisms.

Keywords: clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated-9 (Cas9); hairless; pig model; somatic cell nuclear transfer (SCNT); thymus.

Fig. 1.

Generation and analysis of an hairless (HR)^−/− fetus. (A) T7E1 assay using genomic DNA obtained from an HR^−/− fetus. The arrows indicate the DNA fragments produced following T7E1 digestion. (B) DNA sequences of the HR locus in the HR^−/− fetus. “−” and “+” denote nucleotide deletions and insertions, respectively. (C) Primers used in the T7E1 assay.

Fig. 2.

Western blot analysis of hairless (HR) in eyelid skin samples of HR^−/− and WT piglets. HR was detected using a polyclonal anti-Hr antibody. A polyclonal anti-actin antibody was used as a loading control.

Fig. 3.

Hair loss and accelerated keratinization in hairless (HR)^−/− piglets. (A) Eyelid skin (boxed area) of HR^−/− and WT piglets at postnatal (PN) day 2. (B) Morphological changes in the back skin of HR^−/− piglets. Transmission electron microscopy analysis of the back skin of HR^−/− and WT piglets at the indicated PN day. The circles denote loss of hairs from the follicles. The arrows indicate keratinized stratified squamous epithelium and detachment of the inner root sheath from the hair shaft.

Fig. 4.

mRNA expression of hairless (HR), Wise, AXIN2, Caspase14, MSX2, and FOXN1 in eyelid skin of HR^−/− and WT piglets at postnatal (PN) day 4. Expression of each gene was normalized against that of GAPDH. *P<0.05. Error bars indicate SEM.

Fig. 5.

Histological analysis of apoptotic cells in the thymus of hairless (HR)^−/− and WT piglets at postnatal (PN) day 4. The arrows indicate apoptotic cells.

Fig. 6.

TUNEL and Hoechst staining of apoptotic cells in the thymus of HR^−/− and WT piglets at PN day 4. TUNEL of apoptotic cells (green) marked using arrow heads and DNA was stained by Hoechst 33342 (blue). Scale bar, 50 µm.

Fig. 7.

Flow cytometric analysis of CD4⁺ CD8⁺ lymphocytes in peripheral blood. These percentages are presented in Table 2.

Fig. 8.

mRNA expression of FOXN1 and MSX2 in the thymus of HR^−/− and WT piglets at postnatal (PN) day 4. *P<0.05. Error bars indicate SEM.