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科学通报  2012 

锌指核酸酶介导的高效多位点基因打靶

DOI: 10.1360/972011-1738, PP. 711-719

Keywords: 锌指核酸酶,基因打靶,多位点,同源重组,定点整合,稳定表达,转基因动物,基因治疗

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Abstract:

该研究旨在建立锌指核酸酶介导的多位点基因打靶技术,为获得稳定遗传的转基因动物或基因治疗临床应用解决技术难题.首先利用OPEN平台设计、构建能识别人基因组内rDNA基因间隔序列的锌指蛋白基因序列,与FokⅠ的切割结构域连接、表达后获得锌指核酸酶基因,再构建锌指核酸酶真核表达载体.另外,构建含有2条同源重组引导序列和绿色荧光蛋白基因(EGFP)的多位点基因打靶载体.将锌指核酸酶真核表达载体和多位点基因打靶载体共转染HEK293细胞,内参对照PCR-灰度分析法检测外源基因定点整合效率,结果显示单独转染多位点基因打靶载体的定点整合效率为6.8%;而由于锌指核酸酶在染色质DNA上切割rDNA基因的间隔序列,诱导高效同源重组,锌指核酸酶载体、多位点基因打靶载体共转染的定点整合效率为24.2%,较常规基因打靶定点整合率(10-6~10-5)提高了24000多倍.共转染的HEK293细胞在无任何筛选的条件下持续培养2个月,经过20次传代之后,子代细胞能够持续表达EGFP,提示表达稳定.本研究建立了锌指核酸酶介导的高效多位点基因打靶技术,不仅大大提高了外源基因的定点整合效率,而且兼顾了基因表达的稳定性和安全性,为动物定点转基因和人类基因治疗提供了重要的技术平台,具有广泛的应用前景.

References

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[9]  唐冬生, 刘晋荣, 蒋泓, 等. 大分子BAC 基因打靶载体转染鸡胚原始生殖细胞的研究. 中国组织工程研究与临床康复, 2009, 13:1918-1922
[10]  唐冬生, 刘广振, 刘东军, 等. 奶牛胎儿细胞多位点基因打靶的研究. 佛山科学技术学院学报(自然科学版), 2008, 26: 39-44
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[12]  Beumer K, Bhattacharyya G, Bibikova M, et al. Efficient gene targeting in Drosophila with zinc-finger nucleases. Genetics, 2006, 172:2391-2403
[13]  Bibikova M, Beumer K, Trautman J K, et al. Enhancing gene targeting with designed zinc finger nucleases. Science, 2003, 300: 764
[14]  Morton J, Davis M W, Jorgensen E M, et al. Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells. Proc Natl Acad Sci USA, 2006, 103: 16370-16375
[15]  Foley J E. Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by oligomerized pool engineering. PLoS ONE,2009, 4: 43-48
[16]  Doyon Y, McCammon J M, Miller J C, et al. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol, 2008, 26: 702-708
[17]  Meng X, Noyes M B, Zhu L J, et al. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol, 2008,26: 695-701
[18]  Lloyd A, Plaisier C L, Carroll D, et al. Targeted mutagenesis using zinc-finger nucleases in Arabidopsis. Proc Natl Acad Sci USA, 2005,102: 2232-2237
[19]  Wright D A, Townsend J A, Winfrey R J Jr, et al. High-frequency homologous recomnination in plants mediated by zinc-finger nucleases. Plant J, 2005, 44: 693-705
[20]  Santiago Y, Chan E, Liu P Q, et al. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci USA, 2008, 105: 5809-5814
[21]  Urnov F D, Miller J C, Lee Y L, et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature,2005, 435: 646-651
[22]  Maeder M L, Thibodeau-Beganny S, Jeffry D S, et al. Oligomerized pool engineering (OPEN): An ‘open-source’ protocol for making customized zinc-finger arrays. Nat Protocol, 2009, 4: 1471-1501
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