The Establishment Of SNP Genetic Identification Scheme In Mouse Frozen Embryos And Sperm

Background Currently more than 32000 mouse strains have been established worldwide. To protect the important genetic resource and prevent the loss of mouse resource, embryo and sperm frozen library has been set up to replace the traditional continuous reproduction by America, Europe and Japan. Jackson Laboratory occupies the most abundant resource of mice and the largest frozen embryo library. Recently, National Resource Center(NRLARC) for Rodent Laboratory Animal, National Resource Center of Model Mice(NRCMM) and Frozen Library were established in our country. There are factors that affect the quality of mouse genetic under low temperature, such as the genetic characteristics stability of frozen embryos and sperm, which is critical in the modern biomedical research. The method of individual mouse genetic detection has been developed, while there is still lack of scheme of frozen mouse embryos and sperm genetic test in China, owing to the preciousness and the limit of samples. In addition, the amount of DNA is far lower than normal individual. Therefore, the primary task is developing the infinitesimal DNA pre-treatment and DNA amplification scheme. After the preliminary amplification of DNA samples, the mouse genetic background and strains can be identified.Methods The frozen mouse embryos and sperm come from Chinese Academy of Sciences, Shanghai Experimental Animal Center. After lysis of the mouse embryos, large amounts of DNA was obtained by using whole genome amplification kit and the sperm DNA are gained by using animal genomic DNA rapid extraction kit. DNA is detected by 1% agarose gel electrophoresis.DNA is enriched by using four sets multiplex PCR and the PCR products are used as templates to the reaction of ligase detection reaction. Next, 6% standard denaturing sequencing PAGE gel electrophoresis was used for the detection of LDR products. Last, the software of Gene TM672 is applied to the collection of data and the genotype. The PCR-LDR genotyping panels are constructed for common inbred mice in this paper. 45 sites of SNPs from 21 chromosomes, which are divided into four groups, are selected as detected targets. And primers and probes are specially designed to these SNPs.Results Frozen mouse embryos and sperm DNA extraction scheme. 2 cell stage embryos are get by micro selected method. Next, 200 mol / L KOH, 50 mmol / L dithiothreitol(PH8.3) are added directly to get the early embryo DNA; DTT and protein K are to get sperm DNA. This scheme can efficiently obtain DNA of embryos and sperm.Owing to the rare of the amount of mouse embryo DNA, we use whole genome amplification kit to augment trace DNA. The results showed that the embryonic cell mass is more than or equal to 12 after amplification and detection.The scheme of PCR-LDR genotype identification is suitable for mouse 45 SNPs genotyping. FVB mouse is used as a sample to verify the genotype of 45 SNPs. The result was consistent to information of NCBI database.We successfully apply these schemes to identify 12 strains of mouse frozen embryo and sperm. The results showed that 45 SNPs are all detected and the probability was up to 95%. The NOD, DBA1 strains of mouse embryos have only one SNP that did not detected. And EG, SCID strains have only two SNPs that did not detected. While BALB/C, ACA strains have four SNPs are different with the NCBI database information; Contrasting 12 strains of pairwise, the biggest different point numbers are 30, the minimum different point numbers are 8, the average different point numbers are 19. The mouse sperm NOD, B10 A strains have not detected one SNP, NOD-Scid, Scid-BG strains have not detected two SNPs, C3H-Scid, C3H/ORL strains have four SNPs different with the NCBI database information; Contrasting 12 strains of pairwise, the biggest different point numbers are 29, the minimum different point numbers are 7, the average different point numbers are 18. Thus, 45 SNPs identification effect on mouse strain frozen embryos and sperm are higher, and it is avoiding that only by a few SNPS to be distinguished the strains. In order to verify the ICR, KM is closed colony mouse, we establish 12 strains of embryos and sperm evolutionary tree.Conclusions In this study, mouse frozen embryos and sperm are provided by Shanghai laboratory animal research center. By using direct lysis, whole genome amplification and PCR-LDR method, we successfully established the mouse frozen embryos and sperm 45 SNPs identification.