Abstract: 【Objective】 The objective of this study is to identify 3 transgenic beifers cattle by testing mRNA and protein expression of exogenous FABP4 gene, so as to provide a scientific basis for the further study of the safety of genetically modified cattle. 【Method】 The bovine FABP4 gene was modified, which was obtained from GenBank (accession No. NM_174314), to form an exogenous FABP4 gene by the degeneracy of synonymous codon. Then the pEGFP-C1-FABP4 eukaryotic expression vector was constructed and transferred into recipient cows by somatic cell nuclear transfer and embryo transfer technology. The No.1 and 2 transgenic animals were born on July 19, 2012 and No.3 individual was on August 1, 2012, but No.1 individual was dead on July 20, 2012. The tissue samples of No.1 transgenic individual and blood samples of No.2&3 transgenic individual and Bos Taurus at 8, 10, 12 and, 14 months were collected for identification. The RT-PCR was used to detect whether the transgenic individuals are positive or not. The fluorescence quantitative PCR was used to detect the mRNA expression of the exogenous FABP4 gene in different tissues of No.1 transgenic individual. Expression trend of exogenous, endogenous FABP4 gene of No.2&3 and Bos taurus individuals at 8, 10, 12 and, 14 months and difference of mRNA expression of FABP4 gene between transgenic and non-transgenic animals were analyzed. Moreover, the protein expression of exogenous FABP4 gene in transgenic cattle was detected by Western Blot.【Result】The results showed that three transgenic animals were all positive, because the production of exogenous FABP4 gene, which is 205 bp size, was detected in transgenic cattle, but pregnant cow and negative control didn′t have. The mRNA expressions of exogenous FABP4 gene were observed in different tissues of No.1 transgenic individual. The expressions of this gene in fat tissue and longissimus were significantly higher than other tissues, and it was the lowest in lung. With months changing, exogenous FABP4 gene expression of No.3 transgenic individual was higher than No.2′s and it presented an earlier increase and later decrease trend. The exogenous FABP4 gene expression level of No.2 individual rose from 8th to 12th month, and then it began to decline slightly. However, expression level of No.3 individual rose from 8th to 10th month, and then decreased gradually. The expression levels of endogenous FABP4 gene in transgenic and non-transgenic cattle displayed that it gradually declined by month in non-transgenic animals, but increased in transgenic one, and the expression level of transgenic cattle was lower than non-transgenic animal. It showed that the exogenous FABP4 gene had inhibition effect on the expression of endogenous FABP4 gene. The superposition expressions of exogenous and endogenous FABP4 gene demonstrated that entire FABP4 expression of transgenic individual was greatly higher than non-transgenic one. The result of protein expression was in close agreement with the result of mRNA expression. In addition to the lung, other tissues of No.1 individual all expressed FABP4 protein, and the expression of the longissimus was the highest. The protein expression level of No.3 individual was higher than No.2.【Conclusion】Three FABP4 transgenic animals were successfully acquired and exogenous FABP4 gene could be expressed normally in the transgenic animal′s body. The FABP4 expression of transgenic individual increased greatly than non-transgenic one. The exogenous FABP4 gene inhibits the expression of endogenous FABP4 gene in transgenic individual.

Key words: cattle, transgene, FABP4 gene, intramuscular fat, biological identification