The Study Of Genetic Monitoring In Laboratory Mouse And Rat Strains Through Microsatellites And SNP

The genetic quality of laboratory animals is an important factor that influences the authenticity of the experimental results in the studies that use animals as the main research object.Genetic drift,genetic pollution and other reasons cause the decline of genetic quality during the animal breeding and feeding process.The reduced genetic quality affects the biological characters of laboratory animal and further affects the repeatability and reliability of zoopery.Therefore,we must control genetic quality by using the genetic monitoring so as to ensure the homozygosis and the homogeneity of laboratory animal.We also can eliminate the groups do not conform the criterions and make the correct treatment to the new substrains and mutant strains timely.So the genetic monitoring has very important theoretical and practical significance to ensure the reliability in the results of animal experiments.In present,the reports about genetic monitorings using microsatellite and SNP emphasis emphasize on different aspects.The genetic monitorings for the common rat and mouse strains are not perfect and systematic.So it is necessary to reselect and optimize some markers so that establish a new optimized system for genetic monitoring of the commonly used rat and mouse strains.Further the current gene variation and the forming process of substrains are detected in corresponding strains by the new optimized system.In addition,there is few report about genetic monitoring for the gene modified animal in the world especially in the X mice in present,which possess the highest level of immunodeficiency nowdays.Whether the gene modification will result in the mutations of the genetic monitoring markers,what kind of molecular biological relationship is between the gene modification and the variants of the markers and how to use these markers to monitor the gene modified animal have not been reported.In our present study,we first used microsatellites for genetic monitoring.And we improved the PCR detection conditions in order to establish a stable and reliable monitoring system suits to monitor and identify five mouse strains and three rat strains commonly used.We also hope to do the genetic monitoring for several mouse strains and three rat strains accurately and systematically by the optimized system.Then,we used SNP for genetic monitoring.We screened 32 SNP sites distributed on different chromosomes and monitored 8 mouse strains included five inbred strains,two mutant strains and respective background strains.Through the SNP genotyping assay,we expected to found the degree of variation in inbred strains and the polymorphic sites related with genetic modification in mutant strains.Based on the above results,we obtained the following results:1.An stable and reliable genetic monitoring method was established through screening the database and optimizing the conditions of PCR and agarose gel electrophoresis.A microsatellite-set was also established supporting the method.The set is fit for monitoring of five inbred mouse strains and three inbred rat strains.In addition,we provided the best combination of microsatellites for monitoring of different strains.The detection system is valuable for genetic monitoring as it can fast monitor and identify the related strains accurately.2.Using of the genetic monitoring system,we found that mutant microsatellites distributed in different strains.The variation at these sites can be regarded as the forming process of substrains.3.We chose 24 microsatellites to examined X immunodeficient mice by above detection system.Compared with background mice,the X mice did not appeared mutations at these sites.This indicated that there was no sensitive site related with the gene mutations in 24 microsatellites.4.Using the optimized 32 SNP probes,we made genotyping assay for the 5 inbred mouse strains.Results showed that for the productive groups,BALB/c strain mutated at the sites of rs13483601(A/A?A/G),rs13459176(A/A?A/C)and rs13482131(C/C?C/T);C3H strain mutated at the sites of rs13459176(A/A?A/C)and rs13482131(C/C?C/T);DBA/2 strain mutated at the sites of rs13483295(T/T?G/T);C57BL/6 strain mutated at the sites of rs3653651(C/C?C/T),rs13459176(A/A?A/C)and rs13482131(C/C?C/T);129 strain did not mutated at any of the 30 sites.For the core groups,only rs13459176 site showed heterozygous mutations(A/A?A/C)in C57BL/6,C3 H and BALB/c mice.5.Compared with background mice,we discovered 7 mutant sites included rs13478818,rs13482843,etc.in BALB/c Nude mice by SNP genotyping assay.In X immunodeficienct mice,mutations occurred only at the rs13478215 site(C/C?C/A;C/C?A/A),and the other sites were consistent with the background mice.The polymorphism of these sites indicated that they were more likely to be "hot spot" associated with gene modification.Conclusions:1.The microsatellite-system using for genetic monitoring is stable and reliable.3 inbred rat strains and 5 inbred mouse strains were detected by the genetic monitoring system accurately.We found that some mutations are distributed in these strains.The mutations suggested that these inbred strains gradually divided into substrains or genetic pollution occurred in them.The groups,which were polluted genetically or had undesired mutations,should be promptly eliminated.We also should replenish the eliminated groups by reintroduction or recovery of the cryopreserved embryos for repropagation.These are important to ensure the accuracy and precision of the animal experiments.2.24 microsatellites were detected in X immunodeficienct mice and no variation was discovered.This illustrated that there was no mutations associated with genetic modification in thses 24 microsatellites.In our present study,we first detected the sensitive microsatellites associated with gene mutations in the X immunodeficient mice,and this provided a valuable reference for researching the molecular biological mechanism between the microsatellites and immune gene modifications.3.We detected and analyzed several mouse strains by SNP genotyping experiments.Resuls showed that the amount of mutation sites in 5 inbred core groups was less than production groups.Because the core groups were controlled the progenitive generations strictly and made the progenitive generations lower than the production group.New substrains that will be retained should mate to be homozygous and establish detailed records promptly.4.We discovered 7 mutant sites in BALB/c Nude mice through SNP genotyping experiment.Furthermore,we discovered mutation at the rs13478215 site in X immunodeficient mice for the first time.This result indicated that the total SNP possessed was higher polymorphism than microsatellites and was more sensitive detecting the genetic variation.These mutant sites were more likely to have close relationship with the modified genes.It laid the foundation for clarifying the relationship between SNP variation and gene modification.It also provides a new prospect for genetic monitoring of the new genetically modified animals.