Rapid discrimination of MHC class I and killer cell lectin-like receptor allele variants by high-resolution melt analysis
- 187 Downloads
Ly49G and H-2 class I Dk molecules are critical to natural killer cell-mediated viral control. To examine their contributions in greater depth, we established NK gene complex (NKC)/Ly49 congenic strains and a novel genetic model defined by MHC class I Dk disparity in congenic and transgenic mouse strains. Generation and maintenance of Ly49 and H-2 class I select strains require efficient and reproducible genotyping assays for highly polygenic and polymorphic sequences. Thus, we coupled gene- and allele-specific PCR with high-resolution melt (HRM) analysis to discriminate Ly49g and H-2 class I D and K alleles in select strains and in the F2 and backcross hybrid offspring of different genetic crosses. We show that HRM typing for these critical immune response genes is fast, accurate, and dependable. We further demonstrate that H-2 class I D HRM typing is competent to detect and quantify transgene copy numbers in different mice with distinct genetic backgrounds. Our findings substantiate the utility and practicality of HRM genotyping for highly related genes and alleles, even those belonging to clustered multigene families. Based on these findings, we envision that HRM is capable to interrogate and quantify gene- and allele-specific variations due to differential regulation of gene expression.
KeywordsGenotyping H-2 D Klra7 Ly49g NK gene complex Transgene copy number
This research was supported with Public Health Service grants (AI050072 and AI082024) from the National Institutes of Health, National Institute of Allergy and Infectious Diseases. We thank Jessica Prince, Jack Cronk, Heather Lee, and Thuan Nguyen for technical support.
- Adam SG, Caraux A, Fodil-Cornu N, Loredo-Osti JC, Lesjean-Pottier S, Jaubert J, Bubic I, Jonjic S, Guenet J-L, Vidal SM, Colucci F (2006) Cmv4, a new locus linked to the NK cell gene complex, controls innate resistance to cytomegalovirus in wild-derived mice. J Immunol 176:5478–5485PubMedGoogle Scholar
- Desrosiers M-P, Kielczewska A, Loredo-Osti J-C, Adam SG, Makrigiannis AP, Lemieux S, Pham T, Lodoen M, Morgan K, Lanier LL, Vidal SM (2005) Epistasis between mouse Klra and major histocompatibility complex class I loci is associated with a new mechanism of natural killer cell-mediated innate resistance to cytomegalovirus infection. Nat Genet 37:593–599PubMedCrossRefGoogle Scholar
- Kielczewska A, Pyzik M, Sun T, Krmpotic A, Lodoen MB, Munks MW, Babic M, Hill AB, Koszinowski UH, Jonjic S, Lanier LL, Vidal SM (2009) Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response. J Exp Med 206:515–523PubMedCrossRefGoogle Scholar
- Xie X, Stadnisky MD, Coats ER, Ahmed Rahim MM, Lundgren A, Xu W, Makrigiannis AP, Brown MG (2010) MHC class I D(k) expression in hematopoietic and nonhematopoietic cells confers natural killer cell resistance to murine cytomegalovirus. Proc Natl Acad Sci U S A 107:8754–8759PubMedCrossRefGoogle Scholar