Embryo sexing by loop-mediated isothermal amplification of trace target DNA sequences in cattle and water buffaloes

摘要

PCR, which allows specific amplification of a target DMA sequence, has contributed substantially not only the progress of basic science but also the industrial application of the molecular biology. In the domestic animal breeding, the prediction of the sex of offspring has not been difficult by the emergence of PCR. Briefly, PCR makes it possible to detect the presence of Y-chromosome specific DNA sequence in a few blastomeres removed from preimplantation embryos. This sensitive and reliable technology was combined with the embryo transfer technology to obtain sex predicted offspring. In this study, the author attempted to establish a field applicable em bryo sexing procedure and a sex chromosomal chimerism analysis using a novel DNA amplification method, LAMP, which amplifies a target DNA sequence under isothermal condition. In Chapter 1, a rapid and reliable sexing procedure with LAMP for cattle (Bos taunts) was established. Primer sets for LAMP to amplify bovine male-specific sequence, S4, and male-female common sequence, 1.715 satellite, were designed, respectively. LAMP reaction using the male-specific primer set determined the existence of the Y-chromosome in male embryos. LAMP reaction using the male-female common primer set was used to confirm the absence of blastomeres in sample collection tubes, and thus false judgments of samples (determined to be female) were excluded. In the first experiment, DNA extraction methods for LAMP-based embryo sexing were examined, and heat, NaOH, and PK-TWtreatments showed similar DNA extraction efficiency (88.9 to 94.4% ). The proportion of samples in which the sex was correctly determined was 75 to 100% for 1 to 5 biopsied cells. Lastly, in vivo-derived embryos were examined to verify the usefulness ofLAMP-based embryo sexing, and some of these fresh, sexed embryos were transferred into recipient animals. All calves bora were of the predicted sex (12 male and 21 female). The time needed for sexing was <1 h. Therefore, LAMPbased embryo sexing accurately determined gender and was suitable for field application. In Chapter 2, sex chromosomal chimerism analysis in heterosexual twin female calves was attempted using the cattle embryo sexing procedure with LAMP. When blood samples were diluted 1:1000 in LAMP reaction mixture, hemoglobin or blood coagulation did not influence the turbidity measurement of the reaction mixture for detection of amplified DNA, This procedure could detect the existence of XY leukocytes of 0.01% in female blood. Furthermore, all heterosexual twin female calves (n=10), bearing sex chromosomal chimerism based on karyotyping and PCR, showed male-specific DNA from peripheral blood by LAMP. These results indicated that the cattle embryo sexing procedure with LAMP was available forfreemartin diagnosis based on detection of sex chromosomal chimerism in cattle. In Chapter 3, Y-chromosome-specific sequences (sBuRY.2 and rBuRY.2) in water buffaloes (Bubalus bubalis ) were identified. LAMP reaction using the primer set for sBuRY.2 determined the existence of Y-chromosome in male embryos. LAMP reaction using the primer set for 12S rRNA was used to confirm the absence of blastomeres in sample collection tubes as well as 1.715 satellite in Chapter 1. When fibroblasts were used as the DNAtemplate, DNA samples prepared by NaOH treatment were more suitable for sexing by LAMP than that by heat denaturation. Embryo sexing was also performed using blastomeres from interspecies NT embryos. The sex determined by LAMP for blastomeres corre-sponded with the sex of nuclear donor cells in analyses using >= 4 blastomeres as the templates. The total time for embryo sexing was about 1 h. These results showed that the present procedure without thermal cycling and electrophoresis was reliable and applicable for water buffalo embryos. In conclusion, this study showed that LAMP is very useful for DNA diagnoses using a small amount of templates in the field. The author suppose that LAMP makes farm