TALE技术编辑和调控绵羊成纤维细胞β-酪蛋白基因研究

Researchers have been trying to create an mammalian model of the mammary gland bioreactor by the integration of a single-copy of the exogenous gene into a highly expressed milk protein gene locus in mammalian genome. This approach is likely to yield high levels of protein expression as the foreign gene is surrounded by all the regulatory DNA sequences required for the abundant expression of the replaced milk protein gene. Rapid development TALE (transcription activator-like effector,TALE) technology is becoming an effective tool for genome editing and transcriptional modulation. Tow predetermined sites in the β-casein exon 1 or exon 2 locus are as targeting sites for custom TALENs (TALE nucleases, TALENs) in sheep genome. The activity of TALENs is tested and screened by Surveyor nuclease after electric transfection DNA into embryonic fibroblasts of East Friesian Dairy sheep. Several targeting sites upstream of the transcription start site of the β-casein gene are determined for custom TALE-TFs (TALE transcription factors, TALE-TFs). The activity of TALE-TFs initiating the natural β-casein-promoter exprssion cassette in embryonic fibroblasts of East Friesian Dairy sheep is tested by quantitative reverse-transcription PCR and identified by Northern and Western blot after electric transfection DNA. After screening high activity TALENs and TALE-TFs, a single copy of the red fluorescent protein reporter gene (Red) is integrated into the predetermined targeting sites of the β-casein exon 1 or 2 by homologous recombination using TALENs with an exogenous Red gene donor DNA template. Then the expression result of the formation of the cassette including the natural β-casein-promoter and integrated Red gene is analyzed and identified by TALE-TFs in embryonic fibroblasts. The cells expressing right interesting proteins by TALE-TFs are as donor cells for sheep somatic-cell nuclear transfer. An idea sheep model of the mammary gland bioreactor is constructed by TALE technology combined with somatic-cell nuclear transfer technology. This strategy provides a generic alternative for the expression of foreign proteins in sheep milk. Research purpose is to establish that the expression model of exogenous transgene is modulated by the endogenous regulatory elements of mammalian milk protein gene.

在哺乳动物高表达乳蛋白基因位点，靶向整合外源单拷贝基因，通过内源乳蛋白基因调控机制，实现外源整合基因表达，是研究者一直努力创建的乳腺生物反应器模型。快速发展的TALE技术正在成为基因组编辑和基因转录调控的有效工具。选择东弗里生羊胚胎成纤维细胞，针对β-酪蛋白基因第1或第2外显子位点，利用Surveyor突变检测技术，筛选基因编辑活性高的TALENs。在成纤维细胞内，利用qRT-PCR技术，筛选能够有效激活内源β-酪蛋白基因启动子的TALE-TFs。将高活性的TALENs和TALE-TFs结合，在预先设定的β-酪蛋白基因靶位点，整合外源单拷贝红色荧光蛋白报告基因，形成新的表达框，直接在成纤维细胞激活β-酪蛋白基因启动子，检测外源整合基因的表达结果，筛选正确的核供体细胞，经体细胞核移植克隆技术，建立东弗里生羊乳腺生物反应器，用内源基因的调控元件控制外源整合基因表达。

许多转基因家畜通过乳腺产生治疗性蛋白质。 然而，目的基因整合到基因组的随机位点，蛋白质的表达和分泌会不同。 另外，远端增强子对于基因转录调控和组织特异性表达非常重要。.将人工核酸酶和人工转录因子结合，在预先设定的绵羊ß-酪蛋白基因靶位点，整合外源单拷贝鸡传染性法氏囊VP2抗原蛋白基因，形成新的表达框，直接在成纤维细胞激活ß-酪蛋白基因启动子，检测外源整合基因的表达结果，筛选正确的核供体细胞，经体细胞核移植克隆技术，建立用内源基因的调控元件控制外源整合基因表达的东弗里生羊乳腺生物反应器模型。.针对绵羊β-酪蛋白基因位点，获得人工转录因子（TALE-TFs和SMA），在绵羊成纤维细胞中激活β-酪蛋白基因转录。针对绵羊β-酪蛋白基因第二外显子、第三外显子和第七外显子特定位点，获得人工核酸酶（TALENs和CRISPR-Cas9），在特定位点诱导产生基因组双链断裂。在东福利生绵羊成纤维细胞β-酪蛋白基因第二外显子信号肽位点，同源重组导入鸡传染性法氏囊VP2抗原基因。同源重组成纤维细胞作为核供体，产生克隆的胚胎，用于开发在乳腺中表达VP2蛋白的转基因绵羊。从2015年至今，累计移植受体母羊242只，但未能获得成功出生的羔羊。