Molecular Characterization Of FSHα/βTransgenic-clone Pigs And Detection Of Their Physiological And Biochemical Parameters

China is the largest pork production and consumption in the world. It is particularly important to develop Chinese own excellent commercial pig breeds. The current biotechnology, especially transgenic technology provides novel tools for pig breeding. Reproductive traits are economically important traits, and litter size is an important indicator of reproductivity in pigs. Litter size is a complex trait what was affected by various factors such as ovulation number, conception rate, embryo survival rate and environmental effects. FSHβ is a major gene for litter size in pigs. We have previously produced5FSHα/β transgenic Large White pigs using Erhualian BACs and somatic cell cloning technology.In this study, we performed the systematical investigation of the FSH transgenic pigs, including detection of the Neo marker gene by routine PCR, determination of the copy number and expression level of the exogenous FSH gene by qPCR and Western blot, characterization of the flanking sequences around the inserted sites by TAIL-PCR and Genome Walking. The results showed that the five transgenic pigs are positive transgenic cloned pigs. The copy number of FSHa ranged from11-13, and the number of FSHβ from5to6. Western blot analysis showed that the FSHβ expression levels in brain, ovary and muscle of transgenic piglets are more than4.0folds compared with normal Large White piglets. Neo is expressed in blood of transgenic pigs T30and T32, but not in blood and ovary of Large White pigs. DNA methylation analysis revealed that the transgenic pig has a lower level of methylation in the upstream sequences of FSHβ compared with large White pigs which is consistant with the high expression of FSHβ in transgenic pigs. Using the Genome Walking technology, we detected3integration sites of the exogenous FSHβ gene. One is located at48853791bp on chromosome8,0.7Mb to the upstream gene IGFBP7, and3.3Mb far away from downstream gene PPIC. The second integration site is located at132042158bp on chromosome6, in the intron3of CTH gene,0.5Mb to the upstream gene PTGER3, and38kb far away from downstream gene ANKRD13C. Another integration site is located at160506875bp on chromosome13, in the intron2of CD47gene,0.17Mb to the upstream gene IFT57, and0.25Mb far away from downstream gene BBX. Moreover, we tested the growth and development, blood parameters and sex hormones in the transgenic pigs. The results showed that the growth curve and blood parameters of transgenic pigs had no difference with non-transgenic pigs.This study systematically charaterized the FSHα/β transgenic pigs at the molecular level, and detection and obtained conventional biochemical and physiological indexes of these pigs. The finding paved the load for further purifying selection and evaluation of biological safety of these transgenic pigs.