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

Guanjie Sun,Peiqing Cong,Xiaohong Liu,Yaosheng Chen,Zuyong He,Delin Mo,Yufeng Qin,Xuan Shi,Xinyu Liang,Ke Qin

doi:10.3389/fgene.2020.00138

Abstract

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.

Highlights

Due to domestication and long term selection, dominant white is a widespread coat color among domestic pig breeds, such as Landrace and Yorkshire (Wiseman, 1986)
In order to investigate the effect of increased KIT expression on coat color, we knocked in the CDS of the KIT gene linked with an enhanced green fluorescent protein (EGFP) reporter via a self-cleaving 2A peptide to facilitate its subsequent identification (Figure 1B)
We found that the number of red blood cells (RBCs) and the mean corpuscular hemoglobin concentration (MCHC) decreased slightly in KITD17/+ mice, with hematocrit (HCT) decreasing significantly (Table S3 and Figure S4), implying the possibility of mild anemia, which is similar to the results found in a mouse model with a KIT loss of function mutation (Ruan et al, 2005)

Summary

Introduction

Due to domestication and long term selection, dominant white is a widespread coat color among domestic pig breeds, such as Landrace and Yorkshire (Wiseman, 1986). KIT has a number of potential tyrosine phosphorylation sites, which interact with multiple downstream signaling pathways, including the PI3K, MAPK, and Src family kinase pathways (Roskoski, 2005; Lennartsson and Rönnstrand, 2012). These pathways are involved in the regulation of cell growth, survival, migration, and differentiation (Imokawa, 2004)

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Results

Conclusion