282 IDENTIFICATION OF SRY AND STEROIDOGENIC FACTOR-1 (SF1) GENES IN CANINE XY MALE-TO-FEMALE SEX DEVELOPMENTAL DISORDER

Abstract

Cloning by the process of somatic cell nuclear transfer (SCNT) has been achieved in a variety of mammalian species and has many promising applications. In this process, however, due to reasons beyond current scientific understanding, many results contrary to expectation have also been produced. For instance, abnormal sex development such as demasculinization has been observed in 1 of 6 healthy German shepherd offspring produced with SCNT (1 normal donor (not cloned), 5 cloned but normally developed progenies, 1 cloned sex reversed progeny, and 1 recloned sex reversed progeny from 1 cloned sex reversed progeny). Sex-determining region Y (SRY) is one of the most basic and crucial genes that initiate male sex determination in many mammals. Steroidogenic factor-1 (SF1, NR5A1), which is closely related to SRY, also regulates several genes involved in sex determination. Numerous studies have reported that reduced or deleted SRY gene expression as well as SF1 gene mutations can produced XY sex reversal. To verify the hypothetical association between phenotypic disorder of sex determination and genetic modification by SCNT, we extracted genomic DNA from tissues of normal progeny (not cloned), primary cultured cells of cloned but normally developed progeny, cloned sex reversed progeny, and recloned sex reversed progeny at the age of 1 year and carried out PCR with produced primers based on available SRY and SF1 gene information (SRY gene from AF107021 in GenBank; SF1 gene from ENSCAFG00000023086 in Ensembl). The cloned PCR products were subcloned into T-vector for sequence analysis, which showed no mutation in genetic sequences of SRY and SF1. Taken together, in our case of abnormal sex determination, there was no apparent association between phenotypic sex determination disorder and SRY/SF1 gene mutation. Other sex reversal and related mutation studies have pointed to a wide range of signal networks that include Sox9 and so forth. Further studies should be focused on these other genes in the signal network.