Genetic diagnosis of claudin-16 deficiency and sex determination in bovine preimplantation embryos.

Renal tubular dysplasia is an autosomal recessively inherited disorder in Japanese black cattle that is due to deletion mutations in the claudin-16 gene and causes chronic renal failure and death of affected animals. Here, we report a multiplex-PCR procedure to determine the genotype for claudin-16 deficiency in preimplantation embryos. The presence or absence of the wild-type and mutant allele(s) was precisely detected with the multiplex-PCR using as little as 5 pg of genomic DNA from leukocytes. When biopsied embryo cells were examined for claudin-16 deficiency, 97.2% of genotypes were consistent with the PCR results obtained for the corresponding embryos. In addition, sexing of embryos by PCR was performed using an aliquot of DNA extracted from biopsied embryo cells, and determination of claudin-16 genotype and sex was successfully achieved with an efficiency of 91.7% for claudin-16 genotyping and 83.3% for sexing. The production of a 100-day fetus that was male and homozygous for claudin-16 deficiency, as expected from the analysis of biopsied embryo cells, gave evidence of the reliability and applicability of this procedure for preventing the transmission of this disease and for enabling advances in animal breeding.