原核注射制备转基因小鼠的技术优化  

Optimizing Protocol for the Preparation of Transgenic Mice(Mus musculus) by Pronuclear Injection

作　　者：杨磊[1] 宋丽爽[1] 刘雪霏[1] 白力格 李光鹏[1] 

机构地区：[1]内蒙古大学哺乳动物生殖生物学与生物技术教育部重点实验室,呼和浩特010071

出　　处：《农业生物技术学报》2016年第8期1278-1284,共7页Journal of Agricultural Biotechnology

基　　金：转基因生物新品种培育科技重大专项(No.2014ZX08007-002)

摘　　要：原核注射技术已经是目前制备转基因小鼠（Mus musculus）最有效的手段,但不同实验室所报道的转基因效率差异较大,效率不高仍是该技术的主要缺点。为提高转基因小鼠的制备效率,本研究对转基因小鼠制备过程中的原核注射的多个方面都进行了改进,尤其是在注射针直径、外源基因浓度、单/双原核注射以及胚胎移植等主要步骤上的进行了优化。实验结果表明,不同浓度的外源基因对转基因小鼠的阳性率有很大影响。外源基因为1.00、2.50、3.00和4.00μg/m L浓度时,转基因小鼠阳性率分别0.67%、14.9%、30.6%和21.4%。其中,外源基因浓度在3.00μg/m L极显著高于其他各组（P〈0.01）。采用阳性率最高的3.00μg/m L浓度又进行了单双/原核注射比较,发现转基因阳性率在进行双原核注射组显著高于单原核注射组（24.3%vs.18.3%,P〈0.01）。将显微注射后的2细胞时期胚胎经输卵管移植到0.5、1.5和2.5 d假孕母鼠体内,产仔率分别为68.6%、67.6%和40.1%,转基因阳性率为17.4%、22.5%和10.1%。比较发现,0.5 d与1.5 d代孕母鼠产仔率没有差异,但1.5 d代孕组转基因阳性率（22.5%）显著高于其他组。结果表明,采用尽可能细的注射针、2～4μg/m L的DNA载体浓度、双原核注射、1.5 d的假孕母鼠和正确的胚胎移植操作,是获得高效率转基因小鼠的关键。本研究对从事动物转基因技术研究的实验室提供一些有益的借鉴。The production of transgenic animals is an important tool for experimental and applied biology.Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. At present, the most effective method for the production of transgenic mice is the pronuclear injection technology. Microinjection of DNA into the pronuclei of zygotes is the simplest and most widely used method for generating transgenic（Tg） mice（Mus musculus）. However, it is always associated with random integration of multiple copies of the transgene, resulting in unstable, low, or no transgene expression due to positional effects and/or repeat-induced gene silencing. The main disadvantage of this technique is that the efficiency is not high. In this paper, the injection needle diameter, concentration ofexogenous gene, single-/double- pronuclear microinjection, embryo transfer and other major steps were optimized. The results showed that different concentrations of exogenous gene had a great impact on the positive rate of transgenic mice. When the injection of exogenous gene concentration in 1.00, 2.50, 3.00 and4.00 μg/m L, the positive rates of transgenic mice were 0.67%, 14.9%, 30.6% and 21.4%, respectively. Among them, the exogenous gene concentration at 3.00 μg/m L was significantly higher than that of other groups（P〈0.01）. In the single-/double-pronuclear microinjection test, the concentration of exogenous gene was 3.00 μg/m L. The positive rate of the double nucleus injection group was significantly higher than that of the single injection group（24.3% vs. 18.3%, P〈0.01）. After micro-injection, the 2 cell stage embryo transplantation of0.5 d, 1.5 and 2.5 d, to the pseudopregnant females through the fallopian tube. The birth rates were 68.6%,67.6% and 40.1%, respectively, and the positive rate were 17.4%, 22.5% and 10.1% respectively. In com

关 键 词：显微操作 显微注射 受精卵 原核 转基因 

分 类 号：Q781[生物学—分子生物学]