影响细菌人工染色体转基因小鼠制备效率的因素分析

doi:10.12300/j.issn.1674-5817.2021.032

摘要/Abstract

摘要： 目的比较细菌人工染色体（bacterial artificial chromosome,BAC）转基因小鼠研制中的几个关键因素如实验小鼠遗传背景、BAC质粒注射浓度、BAC质粒片段大小和BAC注射液存放时间等,优化BAC转基因小鼠研制技术。方法采用相同的BAC DNA制备方法,分别选取C57BL/6J（B6）和FVB/N两种实验小鼠,用质量浓度分别为0.75、1.0、1.5和2.0 ng/µL的BAC注射液进行胚胎显微注射,分析不同BAC DNA片段大小和BAC载体DNA制备后至注射前的时间长度,比较小鼠出生率、转基因小鼠阳性率。结果用FVB/N为背景的转基因小鼠阳性率较用C57BL/6J高（P<0.05）。4个BAC质粒注射质量浓度对应的C57BL/6J和FVB/N仔鼠出生率由高到低排序依次为0.75 ng/µL组>1.5 ng/µL组>1.0 ng/µL组>2.0 ng/µL组。4个BAC质粒质量浓度注射时,C57BL/6J转基因小鼠阳性率由高到低排序依次为1.5 ng/µL>1.0 ng/µL>2.0 ng/µL=0.75 ng/µL,FVB/N转基因小鼠阳性率由高到低排序依次为1.5 ng/µL>1.0 ng/µL>2.0 ng/µL>0.75 ng/µL。当BAC片段大小为197 kb时,仔鼠出生率最高,为（22.49±9.41）%;片段大小为99 kb时仔鼠出生率最低,为（13.61±15.65）%;当BAC片段为197 kb时,转基因小鼠阳性率最高,为（13.56±12.88）%,极显著高于114 kb（P<0.01）,显著高于99 kb（P<0.05）。BAC注射液的存放时间与转基因小鼠阳性率并不呈线性关系。结论相较于C57BL/6J,FVB/N背景更适于BAC转基因小鼠的研制。BAC注射液质量浓度为1.5 ng/µL时,阳性转基因小鼠的制备效率最高。BAC片段大小并非阳性转基因小鼠的限制因素。BAC载体DNA制备后1周内完成注射的效果最佳。

关键词: 细菌人工染色体, 转基因小鼠, 胚胎显微注射

Abstract: Objective By comparing some key factors in generating bacterial artificial chromosome (BAC) transgenic mice, such as the background of strains, the concentration of the BAC DNA used for injection, the size of the BAC DNA, and the storage time of the BAC DNA injection, to optimize the generation of BAC transgenic mice. Methods The same procedure of BAC DNA preparation was used with two mouse strains, i.e. C57BL/6J and FVB/N to produce embryos; four concentrations of DNA for injection (0.75, 1.0, 1.5, and 2.0 ng/µL, respectively) were utilized to microinject into the embryos. The size of BAC DNA fragment and the time from BAC preparation to injection were recorded, and the birth rate and positive rate of transgenic mice were compared. Results The positive rate of transgenic mice with FVB/N background was higher than that of C57BL/6J background (P < 0.05). When different concentrations of BAC DNA were adopted, the birth rates of C57BL/6J and FVB/N mice from high to low were 0.75 ng/µL>1.5 ng/µL>1.0 ng/µL>2.0 ng/µL. When the injection concentration was 1.5 ng/µL, the positive rates transgenic mice in C57BL/6J and FVB/N newborns were the highest, while the positive rate was 0 in C57BL/6J newborns when the injection concentration was 0.75 ng/µL and 2.0 ng/µL. When different concentrations of BAC DNA were adopted, the positive rate of transgenic mice in FVB/N newborns from high to low was 1.5 ng/µL>1.0 ng/µL>2.0 ng/µL>0.75 ng/µL. When the BAC fragment size was 197 kb, the mice birth rate was highest at (22.49±9.41)%, and when the fragment size was 99 kb, the birth rate was lowest at (13.61±15.65)%. When the BAC fragment was 197 kb, the transgenic mice had the highest positive rate of (13.56±12.88)%, extremely significantly higher than that for 114 kb (P < 0.01) and significantly higher than that for 99 kb (P < 0.05). The storage time of BAC injection was not linearly associated with the positive rate of transgenic mice. Conclusion Compared with C57BL/6J, mice with FVB/N background appear more suitable for the generation of BAC transgenic mice. When the concentration of BAC DAN injection is 1.5 ng/µL, the positive rate of transgenic mice is the highest. BAC fragment size is not a constraining factor for positive transgenic mice. The highest efficiency is expected when the BAC DNA is injected within one week after preparation.

Key words: Bacterial artificial chromosome, Transgenic mice, Embryo microinjection

中图分类号:

Q95-33
R-332

王芊芊, 王伟, 刘睿, 卫振, 刘冲. 影响细菌人工染色体转基因小鼠制备效率的因素分析[J]. 实验动物与比较医学, 2021, 41(6): 535-542.

WANG Qianqian, WANG Wei, LIU Rui, WEI Zhen, LIU Chong. Factors Affecting Production Efficiency of Bacterial Artificial Chromosome Transgenic Mice[J]. Laboratory Animal and Comparative Medicine, 2021, 41(6): 535-542.

图/表 5

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