学术
基于Cre重组酶体系的鸡卵清蛋白基因打靶载体的构建
张传生[1,2] 杜立新[1]
[1]中国农业科学院畜牧研究所,北京100094 [2]山东农业大学生命科学学院,山东泰安271018
摘 要:
利用胚胎干细胞基因打靶技术制备转基因鸡是研制鸡输卵管反应器的最佳技术路线。为建立基于Cre／loxp系统的鸡卵清蛋白基因（Ovalbumin gene，OV）位点的双交换打靶载体系统，本研究克隆了鸡的OV基因7．8kb片段，并与克隆的内部核糖体进入位点（IRES）、人工合成的含有Cre重组酶识别位点变异体交换盒m2／loxp71-EGFP-loxp66，一起构建了含有Hsv-tk负筛选标记的针对鸡卵清蛋白基因位点的敲入型共表达基因打靶载体pSSC—m2／71EGFP661RES—OV7．8；以猪β干扰素基因（βInterferon，IFN—β）为目的基因构建了穿梭载体pMD—m2／66-MCS-IFN-MCS-Loxp71，经过限制酶酶切及部分测序鉴定，所构建载体结构正确。进一步将它们共转化组成性表达Cre的细菌BM25．8，验证了loxp突变位点对重组反应的有效性[动物学报51（4）：685—690，2005]。[著者文摘]
文章出处:
《动物学报》-2005年51卷4期 -685-690页
栏目信息:
Construction of chicken ovalbumin gene targeting vector containing the Cre recombinase recognition site
Zhang ChuanSheng;Du LiXin
Abstract:
Gene targeting combined with culture of chicken embryonic stem cells is one of the best strategies to produce chicken oviduct bioreactor. To study the construction of a vector with Cre recombinase recognition site loxp mutant for chicken Ovalbumin gene targeting in embryonic stem cells, the 1.7 kb fragment including chicken partly gene 5＇flanking sequence, and exon 1, intronl and partly exon 2 and the 6.1 kb fragement including OV gene from exon 2 to exon 8 were cloned. Using two chicken OV gene DNA fragments, the internal ribosome entry site （IRES） of the （ECMV） virus and synthesized oligonucleotides, pSSC-m2/71EGFP661RES-OV7.8 was designed and constructed as a gene targeting vector. Then the shuttle vector pMD-m2/66-MCS-IFN-MCS-Loxp71 was constructed. Structures of the two constructs were identified by restrictive digestion analysis and partial sequencing. To check the function of two Cre “exchange cassette” , the targeting construct and the lineal shutter construct were tested in E. coli BM25.8 that express the Cre recombinase constitutively. The results indicated that this targeting construct system can be used in the study of chicken oviduct bioreactor [Acta Zoologica Sinica 51 （4）： 685-690, 2005].[著者文摘]
Key words:
Gene targeting, ES cell, OV, Cre recombinase, Recombinase mediated cassette exchange
基金资助:
北京市自然科学基金资助项目（No.5051004）;致谢 感谢第二军医大学傅继梁教授、第四军医大学孙凯教授和美国Colorado大学Stever Langer博士提供相关实验材料与文献.感谢姜运良教授对本文提出的宝贵意见.
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