泌乳相关染色质开放位点的鉴定及其对外源基因敲入效率和表达的影响

Goat mammary gland bioreactor has unique advantages and wide application prospect for large-scale production of exogenous recombinant protein with bioactivity. However, exogenous gene integration is inefficient, and the expression quantity of inserted genes is susceptible to the neighboring chromatin structure which severely plague the research and promotion of this technique. Our previous studies showed that chromatin structure affects the recognition and binding of Cas9 to the livestock genome DNA. Besides, the insertion rate in active locus was generally higher than the inactive locus for the same exogenous gene. On the basis of these findings, this project will obtain several lactation related chromatin accessible target sites through ATAC-seq on the mammary epithelial cells at the lactation and non-lactation period. Furthermore, CRISPR/Cas9 system will be used to explore the influence of chromatin accessibility at target site to the nuclease activity, genome-wide off-target effects, knock-in efficiency, and the expression quantity of inserted gene. Thus, the relevance between chromatin accessibility at target site and the Cas9 induced DNA double strand break and further homologous directed repair will be clarified. These results will be favor to improve the gene target system in livestock, and provide theoretical support for enhancing the efficiency of transgenic goat mammary gland bioreactor production.

山羊乳腺生物反应器对于大规模生产具有生物活性的重组蛋白具有独特优势与广阔应用前景。然而，外源基因整合效率低下且插入基因的表达易受附近染色质结构的影响，严重限制了该技术的研究推广。前期研究表明染色质结构会影响Cas9蛋白对家畜基因组DNA的结合，且外源基因在活跃表达的基因间区域的插入效率一般高于非活跃区域。因此，本项目拟通过对泌乳期和干奶期山羊乳腺上皮细胞进行ATAC-seq，鉴定出多个泌乳相关的染色质开放性打靶位点，并进一步利用CRISPR/Cas9系统，探究泌乳相关染色质开放位点对于Cas9核酸酶活性、全基因组脱靶效应、外源基因敲入效率和插入基因乳腺特异性表达量的影响。进而揭示打靶位点染色质可接近性与同源重组修复途径及外源基因稳定高效表达间的相关性，为改进家畜基因打靶系统，提高转基因羊乳腺生物反应器制备效率提供理论支持。

乳腺生物反应器是生产具有生物活性的重组蛋白的理想反应器，具有广阔的应用前景，其关键在于外源基因的高水平表达和稳定整合。该项目描述了一种鉴定外源基因整合位点以增强转基因表达的方法。结合ATAC-seq和RNA-seq来筛选奶山羊泌乳期乳腺上皮细胞染色质特异性开放的区域，并利用CRISPR/Cas9将人溶菌酶基因整合到4个不同的开放区域。通过比较在不同打靶位点插入基因的表达水平，筛选出有利于转基因表达的整合位点。研究结果表明，打靶位点的染色质开放性与基因插入效率成正相关，且将外源基因整合到宿主细胞高开放的染色质区域可以有效提高转基因的表达水平。在此基础上，利用HMEJ介导的同源重组修复以及内源性启动子启动筛选标记的方法，极大地提高了外源基因的插入效率。该研究将为改进家畜基因打靶系统，提高转基因羊乳腺生物反应器制备效率提供技术支持。

内容获取失败，请点击重试