Production of apolipoprotein C-III knockout rabbits using zinc finger nucleases.
Production of apolipoprotein C-III knockout rabbits using zinc finger nucleases.
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DOI：
10.3791/50957
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发表时间：
2013-11-18
期刊：
影响因子：
--
通讯作者：
Chen YE
中科院分区：
文献类型：
--
作者：
Yang D;Zhang J;Xu J;Zhu T;Fan Y;Fan J;Chen YE
载脂蛋白(Apo)C-III(APOCIII)存在于血浆乳糜粒(CM)、极低密度脂蛋白(VLDL)和高密度脂蛋白(HDL)的表面。现已认识到高水平的血浆载脂蛋白III是心血管疾病(CVD)的危险因素。血浆载脂蛋白III水平升高通常与胰岛素抵抗、肥胖和高甘油三酯血症相关。从包括APOCIII基因敲除(KO)小鼠在内的转基因小鼠模型中，已经获得了关于ApoCIII在脂代谢和心血管疾病中作用的宝贵知识；然而，人们注意到，小鼠的脂蛋白代谢在许多方面与人类不同。直到现在还不知道血浆载脂蛋白III升高是否直接导致动脉粥样硬化。在本研究中，我们致力于开发APOCIII KO兔，基于兔可以作为研究人类脂代谢和动脉粥样硬化的合理模型的假设。针对兔ApoCIII基因的锌指核酸酶(ZFN)组在胚胎显微注射前进行了体外验证。将该基因注射到35个兔原核期胚胎的细胞质中，检测囊胚期的突变率。在检测的16个囊胚中，在靶点获得了令人满意的50%的突变率(8/16)，支持将SET 1用于体内实验。接下来，我们显微注射了145个胚胎，并将这些胚胎移植到7只受体兔身上。妊娠30天后，共产仔21只，其中5只经测序证实为APOCIII KO兔。KO动物率(KO套数/总产仔数)为23.8%。整体生产效率为3.4%(每145个胚胎移植5个试剂盒)。本研究结果表明，ZFN是一种高效生产KO兔的方法。这些APOCIII KO兔是研究APOCIII在脂质代谢中作用的新资源。
Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins (HDL). It has been recognized that high levels of plasma ApoCIII constitutea risk factor for cardiovascular diseases (CVD). Elevated plasma ApoCIII level often correlates with insulin resistance, obesity, and hypertriglyceridemia. Invaluable knowledge on the roles of ApoCIIIin lipid metabolisms and CVD has been obtained from transgenic mouse models including ApoCIII knockout (KO) mice; however, it is noted that the metabolism of lipoprotein in mice is different from that of humans in many aspects. It is not known until now whether elevated plasma ApoCIII is directly atherogenic. We worked to develop ApoCIII KO rabbits in the present study based on the hypothesis that rabbits can serve as a reasonablemodelfor studying human lipid metabolism and atherosclerosis. Zinc finger nuclease (ZFN) sets targeting rabbit ApoCIIIgene were subjected to in vitro validation prior to embryo microinjection. The mRNA was injected to the cytoplasm of 35 rabbit pronuclear stage embryos, and evaluated the mutation rates at the blastocyst state. Of sixteen blastocysts that were assayed, a satisfactory 50% mutation rate (8/16) at the targeting site was achieved, supporting the use of Set 1 for in vivo experiments. Next, we microinjected 145 embryos with Set 1 mRNA, and transferred these embryos to 7 recipient rabbits. After 30 days gestation, 21 kits were born, out of which five were confirmed as ApoCIII KO rabbits after PCR sequencing assays. The KO animal rate (#KO kits/total born) was 23.8%. The overall production efficiency is 3.4% (5 kits/145 embryos transferred). The present work demonstrated that ZFN is a highly efficient method to produce KO rabbits. These ApoCIII KO rabbits are novel resources to study the roles of ApoCIII in lipid metabolisms.
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影响因子：
15.9
作者：
GINSBERG, HN;LE, NA;BROWN, WV
通讯作者：
BROWN, WV
DOI：
10.1126/science.1161524
发表时间：
2008-12-12
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Pollin TI;Damcott CM;Shen H;Ott SH;Shelton J;Horenstein RB;Post W;McLenithan JC;Bielak LF;Peyser PA;Mitchell BD;Miller M;O'Connell JR;Shuldiner AR
通讯作者：
Shuldiner AR
DOI：
10.1073/pnas.1106422108
发表时间：
2011-07-19
影响因子：
11.1
作者：
Hauschild, Janet;Petersen, Bjoern;Niemann, Heiner
通讯作者：
Niemann, Heiner
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
3.3
作者：
Carbery, Iara D.;Ji, Diana;Cui, Xiaoxia
通讯作者：
Cui, Xiaoxia