Inbred Genetic Method For Detection Of The Department Of Rat Major Histocompatibility Complex (rt1)

It becomes more and more necessary for routine monitoring of inbred strains ofrats to ensure their genetic integrity along with the development of biological science.But the existing monitoring methods can't meet the need of that. The majorhistocompatibility of rat (RT1) is the most major gene cluster to decide the quality ofimmunogenetics of rats and also very important molecular genetic marks in thegenetic monitoring of inbred strains of rats. There were few reports about inbredstrains of rats monitored by RT 1 at present. The purpose of our research is to establishthe methods based on serology and molecular biology to improve the geneticmonitoring technique of inbred rat strains.Microcytotoxicity test was set up in the first part of the paper. Firstly, thestrain-specific typing antisera (SSTA) were prepared for three inbred strains of rats byusing pooled immunization protocol. SSTA could detect ClassⅠhistocompatibilityantigens and differentiation antigens of RT6 and RT7. It was concluded that SSTAcould be used in the microcytotoxicity assay to provide with high sensitivity andreproducibility. Secondly, splenic lymphocyte were prepared for microcytotoxicityassay. Cells were counted with a haemocytometer to a final suspension of 2×10~6cells/ml. Viability, as determined by trypan blue staining, was always greater than 95%. Ultimately, complement-mediated microcytotoxicity testing was performed byobserving the percentage of the dead cells over the total cells under microscope.Results of the experiments demonstrated that using SSTA in complement-mediatedmicrocytotoxicity testing is a simple, reliable and repeatable method for themonitoring of inbred strains of rats.The second part of paper is to establish the molecular biological methods tomonitor RT1 haplotype. Firstly, two pairs of general primers were designed to aimdirectly at the strain-specificity regions of RT1Ba and RT1Db, which were comparedand analyzed by Megalin process, according to the primer designing principle.Approximately 742 bp RT1Ba were amplified from BN/CrlBR(RT1~n), but 764 bp RT1Ba fragments from LEW/Cr1BR(RT1~1), F344/CrCr1BR(RT1~1) andSHR/NCr1BR(RT1~k). While 384 bp RT1Db fragments could be amplified from all thefour stains of BN/Cr1BR(RT1~n), LEW/Cr1BR(RT1~1), F344/CrCr1BR(RT1~1) andSHR/NCr1BR(RT1~k). The amplified fragments were sequenced and proved to beconsistent with the corresponding sequence. According to the difference of theamplified fragments, inbred strains of rats were detected and classified by various ofmolecular biological methods. The study not only provided effective experiment datafor genetic identification and distinguishing of rats strains, but also offered moreaccurate genetic background information for genetic monitoring to the inbred strainsof rats.In our research, the difference of RT1 haplotype of inbred rat strains weredetected using molecular biological methods and the strains were distinguished atlevel of serology. Our aim is to search for a more precise genetic monitoring methodfor inbred strains of rats to meet the development of modern biology.