K Locus Effects in Gray Wolves: Experimental Assessment of TLR3 Signaling and the Gene Expression Response to Canine Distemper Virus. Author Rachel Johnston, James Rheinwald, Bridgett vonHoldt, Daniel Stahler, William Lowry, Jenny Tung, Robert Wayne Publication Year 2021 Type Journal Article Abstract In North American gray wolves, black coat color is dominantly inherited via a 3 base pair coding deletion in the canine beta defensin 3 (CBD103) gene. This 3 base pair deletion, called the KB allele, was introduced through hybridization with dogs and subsequently underwent a selective sweep that increased its frequency in wild wolves. Despite apparent positive selection, KBB wolves have lower fitness than wolves with the KyB genotype, even though the 2 genotypes show no observable differences in black coat color. Thus, the KB allele is thought to have pleiotropic effects on as-yet unknown phenotypes. Given the role of skin-expressed CBD103 in innate immunity, we hypothesized that the KB allele influences the keratinocyte gene expression response to TLR3 pathway stimulation and/or infection by canine distemper virus (CDV). To test this hypothesis, we developed a panel of primary epidermal keratinocyte cell cultures from 24 wild North American gray wolves of both Kyy and KyB genotypes. In addition, we generated an immortalized Kyy line and used CRISPR/Cas9 editing to produce a KyB line on the same genetic background. We assessed the transcriptome-wide responses of wolf keratinocytes to the TLR3 agonist polyinosinic:polycytidylic acid (polyI:C), and to live CDV. K locus genotype did not predict the transcriptional response to either challenge, suggesting that variation in the gene expression response does not explain pleiotropic effects of the KB allele on fitness. This study supports the feasibility of using cell culture methods to investigate the phenotypic effects of naturally occurring genetic variation in wild mammals. Keywords Animals, Gene Expression, Alleles, Wolves, Dogs, Distemper Virus, Canine, Toll-Like Receptor 3 Journal J Hered Volume 112 Issue 5 Pages 458-468 Date Published 2021 Aug 25 ISSN Number 1465-7333 DOI 10.1093/jhered/esab029 Alternate Journal J Hered PMCID PMC8567826 PMID 34132805 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML