Genetic monitoring of immunegentic class-I major histocompatibility complex loci in BALB/c mice

Genetic quality control test is one of most important procedures in laboratory animal breeding. This study was aimed to monitor two immunogenic loci, H-2D and H-2K, in BALB/c inbred mouse strain. In this study, according to the FELASA recommendations, a total of 30 mice (15 male and 15 female) were randomly selected from the BALB/c colony. The animals were euthanized by Ketamine-Rampon, and the spleen tissue samples collected under sterile condition. Thereafter, total RNA was extracted and cDNA was synthezed using specific kites. In the current study, two DNA segments of 295 and 820 pb from H-2K and H-2D loci were amplified using specific primers and PCR method, respectively. The nucleoid differences between the sequenced samples were considered to identify alleles and haplotypes. The nucleoid sequences were determined using Sanger method and the primed sequences were then aligned and compared with the standard sequence of the BALB/c sequence as the reference. Results showed that 25 mice of 30 sampled mice had d allele in H-2K loci. In addition, four mice had sequences associated with k allele and one mouse showed mutant-d allele. Also, 25 mice of the 30 sampled mice showed complete identity with the d allele as the reference of H-2D BALB/c allele. However, three mice showed mutant-d allele, while the sequence of two mice did not show identity with d alleles. Moreover, among 30 investigated mice, 25 mice showed standard d haplotype of BALB/c strain. There was no significant difference between male and female mice regarding genetic contamination (P<0.05). These finding show that about 17% genetic contamination in the H-2K and H-2D loci of BALB/c colony which is probably related to single nucleotide mutation or uncontrolled mating. In conclusion, the results of the present study showed that approximately 83% of the BALB/c mice colonies were pure in H-2K and H-2D immunogenic loci.