摘要
目的:确定提高VASN基因敲除小鼠基因型检出率的方法。方法:根据VASN基因外显子设计2对gRNA,将Cas9和gRNA共注射入受精卵生产出F0代小鼠,将F0代小鼠进行近交繁殖产生F1代,分别用3种方法鉴定F1代小鼠基因型,比较3种方法的检出率。方法一:DNA提取试剂盒A+DNA高保真聚合酶a+PCR扩增条件1,鉴定16只小鼠;方法二:DNA提取试剂盒A+DNA高保真聚合酶b+PCR扩增条件2,鉴定14只小鼠;方法三:DNA提取试剂盒B+DNA高保真聚合酶b+PCR扩增条件3,鉴定9只小鼠。结果:Cas9和gRNA共注射入319枚受精卵,分5批移植,其中3批受孕成功,胚胎移植率为60.00%,共获得16只F0代小鼠,经过自交繁殖共获得39只F1代小鼠。分3批分别采用3种方法进行鉴定,方法一的基因型检出率为12.50%;方法二的基因型检出率为64.29%;方法三的基因型检出率为100.00%,且与测序结果吻合,电泳图无杂带。结论:DNA提取试剂盒B+DNA高保真聚合酶b+PCR扩增条件3的基因型鉴定检出率最高。
Objective: To determine the method to improve the detection rate of genotype of VASN gene knockout mice. Methods: Two pairs of gRNA were designed according to VASN gene exons, Cas9 and gRNA were coinjected into fertilized eggs to produce F0 generation mice, and F0 generation mice were inbred to produce F1 generation. Genotypes of F1 generation mice were identified by three methods respectively, and the detection rates of the three methods were compared. Method 1: DNA extraction kit A+DNA high-fidelity polymerase A+PCR amplification condition 1, 16 mice were identified;Method 2: DNA extraction kit A + DNA high-fidelity polymerase B +PCR amplification condition 2, identification of 14 mice;Method 3: DNA extraction kit B+DNA high-fidelity polymerase B+ PCR amplification condition 3, identified 9 mice. Results: Cas9 and gRNA were injected into a total of 319 fertilized eggs and transplanted in 5 batches, among which 3 batches were successfully fertilized, and the embryo transfer rate was 60.00%. A total of 16 F0 generation mice were obtained, and 39 F1 generation mice were obtained through self-cross-breeding. Three genotypes were identified in three batches, and the genotype detection rate of method 1 was 12.50%. The genotype detection rate of method 2 was 64.29%.The genotype detection rate of methods 3 was 100.00%, and the results were consistent with the sequencing results.Conclusion: DNA extraction kit B + DNA high fidelity polymerase B + PCR amplification condition 3 had the highest genotype identification and detection rate.
作者
薛康宁
张名媛
杨丽超
郭晓萍
莫忠香
欧阳轶强
孙俊铭
XueKangning;ZhangMingyuan;Yang Lichao;Guo Xiaoping;Mo Zhongxiang;Sun Junming(Laboratory Animal Center,Guangxi Medical University,Nanning 530021,China;School of Public Health,Guangxi Medical University,Nanning 530021,China)
出处
《广西医科大学学报》
CAS
2021年第2期398-402,共5页
Journal of Guangxi Medical University
基金
中央引导地方科技发展专项(No.桂科ZY1949025)。
关键词
VASN
基因敲除小鼠
鉴定
VASN
knockout mouse
genotype identification
