应用TALEN技术敲除idh1/2基因建立斑马鱼白血病模型
首发时间:2017-05-09
摘要:目的应用TALEN技术,人工构建TALEN 靶向敲除载体敲除idh1/2基因,建立斑马鱼白血病模型。方法采用单元组装法构建靶向敲除idh1/2基因的载体,将其体外转录成TALEN mRNAs,通过显微注射的方式注入单细胞期受精卵中,胚胎期检测TALEN的敲除效率,再通过剪尾、PCR、酶切、测序筛选发生基因突变的斑马鱼及突变的类型。结果酶切、测序结果证实我们成功构建出正确的idh1/2靶向敲除载体,注入斑马鱼受精卵后,基因敲除的效率达到50%以上。通过剪尾、PCR、酶切检测成功地筛选出了发生基因突变的斑马鱼,测序结果显示出不同种类的idh1/2基因突变。结论我们成功构建出idh1/2基因敲除的斑马鱼模型,为进一步研究人类IDH1/2基因突变引起的白血病发病机制提供了良好的动物模型。
关键词: TALEN idh1/2基因 斑马鱼 动物模型 白血病
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Establishment of idh1/2 genes knockout zebrafish leukemia model by TALEN
Abstract:Objective By introducing transcription activator-like effector nuclease (TALEN) vectors,we established idh1/2 genes knockout zebrafish leukemia model. Method Using unit assembly method,We construct TALEN vectors targeting zebrafish idh1/2 genes. The TALEN mRNA was in vitro-transcribed and then microinjected into zebrafish embryos at one-cell stage.The efficiency of TALEN was tested first by injected embryos. Next through PCR and enzyme digestion, the different types of mutations were screened and confirmed. Results According to enzyme digestion and sequencing, we successfully constructed correct targeEstablishment of idh1/2 genes knockout zebrafish leukemia model by TALENting vectors, and the knockout efficiency of idh1/2 was above 50%. We screened the mutant zebrafish through tail-cutting, PCR, Establishment of idh1/2 genes knockout zebrafish leukemia model by TALENenzyme digestion and sequencing, finally,we confirmed different types of idh1/2 genes mutations in injected zebrafish. Conclusions We successfully constructed idh1/2 knockout zebrafish models, which will serve as animals models for further research of human leukemia induced by IDH1/2 mutations.
Keywords: TALEN idh1/2 genes Zebrafish Animal model leukemia
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