Functional characterization of cAMP signaling of variant porcine MC1R alleles in PK15 cells.

The melanocortin 1 receptor (MC1R), encoded by the classical extension (E) coat color locus, is expressed on the surface of melanocytes and plays a critical role in switching melanin synthesis from pheomelanin (red/yellow) to eumelanin (black/brown). Different MC1R alleles associated with various coat color patterns in pigs have been identified over the past decades. However, functional analysis of variant porcine MC1R alleles has not yet been performed. Therefore, in this study, we examined the subcellular localization and cyclic adenosine monophosphate (cAMP) signaling capability of MC1R variants in porcine kidney epithelial cells (PK15) overexpressing different MC1R alleles. Transcriptional slippage may partially restore the reading frame of the EP allele, possibly accounting for the observed spot phenotype. The A243T substitution in the e allele severely disrupted the membrane localization of the MC1R receptor, resulting in a severely impaired cAMP signaling capability. Both the V95M and L102P substitutions in the ED1 allele may contribute to the constitutively active function of MC1R, thus accounting for the dominant black phenotype. The D124N substitution in the ED2 allele severely attenuated the cAMP signaling capability of MC1R; however, whether this mutation contributes to the distinct phenotype of Hampshire pigs requires further investigation. Thus, our results provide new insights into the functional characteristics of MC1R variants and their roles in porcine coat color formation.

Impact of KIT Editing on Coat Pigmentation and Fresh Meat Color in Yorkshire Pigs.

The white coat color of Yorkshire pigs is caused by the dominant white I allele, which has been associated with at least one copy of the 450-kb duplication encompassing the entire KIT gene and a splice mutation (G > A) at the first base of intron 17. The splice mutation in KIT has an adverse effect on pigmentation in mice. Therefore, removing the 450 kb duplications harboring the KIT copy with splice mutations is expected to affect Yorkshire pig pigmentation. In this study, we describe the use of a Yorkshire pig kidney cell strain with the I?/IBe-ed genotype, previously created by CRISPR-Cas9, as donor cells for somatic cell nuclear transfer to generate gene-edited Yorkshire pigs. The removal of the 450 kb duplications harboring the KIT copy with splice mutation did not alter the white coat color of Yorkshire pigs, which was confirmed by the absence of fully mature melanocytes and melanin accumulation in the hair follicles. Except for the improved transcription of tyrosinase, and slight increase in microphthalmia transcription factor and tyrosinase-related protein 1 protein expression, there was no significant impact of the removal of splice mutations on genes and signaling pathways (PI3K/AKT) involved in melanogenesis. However, the removal of the 450 kb duplications harboring the KIT copy with splice mutation substantially improved fresh meat color accompanied by significantly increased red blood cell number, which merits further investigation. Our study provides new insights into the role of structural mutations of the KIT gene in the formation of white coat color and erythropoiesis in Yorkshire pigs.

Functional Analysis of KIT Gene Structural Mutations Causing the Porcine Dominant White Phenotype Using Genome Edited Mouse Models.

The dominant white phenotype in pigs is thought to be mainly due to a structural mutation in the KIT gene, a splice mutation (G > A) at the first base in intron 17 which leads to the deletion of exon 17 in the mature KIT mRNA. However, this hypothesis has not yet been validated by functional studies. Here, we created two mouse models, KIT D17/+ to mimic the splice mutation, and KIT Dup/+ to partially mimic the duplication mutation of KIT gene in dominant white pigs using CRISPR/Cas9 technology. We found that the splice mutation homozygote is lethal and the heterozygous mice have a piebald coat. Slightly increased expression of KIT in KIT Dup/+ mice did not confer the patched phenotype and had no obvious impact on coat color. Interestingly, the combination of these two mutations reduced the phosphorylation of PI3K and MAPK pathway associated proteins, which may be related to the impaired migration of melanoblasts observed during embryonic development that eventually leads to the dominant white phenotype.

Highly efficient correction of structural mutations of 450 kb KIT locus in kidney cells of Yorkshire pig by CRISPR/Cas9.

The white coat colour of Yorkshire and Landrace pig breeds is caused by the dominant white I allele of KIT, associated with 450-kb duplications and a splice mutation (G > A) at the first base in intron 17. To test whether genome editing can be employed to correct this structural mutation, and to investigate the role of KIT in the control of porcine coat colour, we designed sgRNAs targeting either intron 16 or intron 17 of KIT, and transfected Cas9/sgRNA co-expression plasmids into the kidney cells of Yorkshire pigs. The copy number of KIT was reduced by about 13%, suggesting the possibility of obtaining cells with corrected structural mutations of the KIT locus. Using single cell cloning, from 24 successfully expanded single cell clones derived from cells transfected with sgRNA targeting at intron 17, we obtained 3 clones with a single copy of KIT without the splice mutation. Taken together, the 12.5% (3/24) efficiency of correction of structural mutations of 450 kb fragments is highly efficient, providing a solid basis for the generation of genome edited Yorkshire pigs with a normal KIT locus. This provides an insight into the underlying genetic mechanisms of porcine coat colour.

Bone Morphogenetic Protein 15 Knockdown Inhibits Porcine Ovarian Follicular Development and Ovulation.

Bone morphogenetic protein 15 (BMP15) is strongly associated with animal reproduction and woman reproductive disease. As a multifunctional oocyte-specific secret factor, BMP15 controls female fertility and follicular development in both species-specific and dosage-sensitive manners. Previous studies found that BMP15 played a critical role in follicular development and ovulation rate in mono-ovulatory mammalian species, especially in sheep and human, but study on knockout mouse model implied that BMP15 possibly has minimal impact on female fertility of poly-ovulatory species. However, this needs to be validated in other poly-ovulatory species. To investigate the regulatory role of BMP15 on porcine female fertility, we generated a BMP15-knockdown pig model through somatic nuclear transfer technology. The BMP15-knockdown gilts showed markedly reduced fertility accompanied by phenotype of dysplastic ovaries containing significantly declined number of follicles, increased number of abnormal follicles, and abnormally enlarged antral follicles resulting in disordered ovulation, which is remarkably different from the unchanged fertility observed in BMP15 knockout mice. Molecular and transcriptome analysis revealed that the knockdown of BMP15 significantly affected both granulosa cells (GCs) and oocytes development, including suppression of cell proliferation, differentiation, and follicle stimulating hormone receptor (Fshr) expression, leading to premature luteinization and reduced estradiol (E2) production in GCs, and simultaneously decreased quality and meiotic maturation of oocyte. Our results provide in vivo evidence of the essential role of BMP15 in porcine ovarian and follicular development, and new insight into the complicated regulatory function of BMP15 in female fertility of poly-ovulatory species.