FABP4转基因牛的分子生物学鉴定

【目的】对前期获得的3头转基因牛进行分子生物学评价，包括转基因阳性鉴定、基因及蛋白表达水平的检测，旨在进一步为转基因牛的安全评价提供一些科学依据。【方法】根据GenBank上公布的牛FABP4基因（GenBank登录号：NM_174314）mRNA序列，利用兼并密码子对现有FABP4基因进行突变，并构建了pEGFP-C1-FABP4真核表达载体、通过细胞转染、体细胞核移植、胚胎移植技术获得3头转基因牛；分别采集了1号转基因牛不同组织样，2、3号转基因牛与野生型个体8、10、12、14月龄的血液样本，利用RT-PCR技术对所获转基因个体的阳性与否进行了鉴定；同时利用荧光定量PCR技术检测了1号转基因牛不同组织中外源性FABP4基因的表达情况，并分析了2、3号转基因牛在8-14月龄之间，外源性FABP4基因、内源性FABP4基因的变化趋势，并通过叠加转基因牛外源与内源性FABP4基因的表达量，分析了转基因牛和非转基因牛FABP4基因的整体表达水平差异；通过Western blotting技术分析了3头转基因牛外源性FABP4基因的蛋白表达水平。【结果】①对所获得的3头转基因牛RT-PCR检测发现，3头转基因个体各样本均在205 bp处产生条带，而妊娠母牛及阴性对照在205 bp处均未有条带出现，初步表明3头转基因牛均为转基因阳性。②实时定量PCR检测表明，1号转基因牛的外源性FABP4基因在多数组织均有较高表达，其中脂肪组织表达量最高、肾脏和背最长肌次之，肺部的相对表达量极低。3号转基因牛外源性FABP4基因的表达水平高于2号转基因牛，且2、3号转基因牛外源性FABP4基因的表达水平随月龄变化均呈现先上升后下降趋势；在内源性FABP4基因表达水平检测中发现，外源性FABP4基因的转入抑制了转基因牛体内内源性FABP4基因的表达，且随月龄变化转基因牛体内内源性FABP4基因表达水平均呈现上升趋势；野生型牛内源性FABP4基因的表达水平随着月龄变化逐步下降，而转基因牛体内内源性FABP4基因的表达量均低于野生型牛；通过对2、3号转基因牛外源与内源性FABP4基因相对表达量的叠加分析发现，转基因牛FABP4基因整体表达量较野生型大幅升高。③蛋白表达检测结果与mRNA检测结果基本一致，外源性FABP4基因在1号转基因牛除肺部外的各个组织均检测到外源FABP4基因的蛋白表达，其中背最长肌表达量最高；3号转基因牛蛋白表达水平高于2号转基因牛。【结论】获得的3头牛均为FABP4阳性转基因牛，且外源性FABP4基因在体内能够正常表达，转基因牛FABP4表达量较野生型大幅升高，同时外源性FABP4基因的转入抑制了内源性FABP4基因的表达。

The Biological Identification of FABP4 Transgenic Cattle

【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.