食管癌细胞Eca9706 DNA聚合酶β基因敲除载体的构建

Construction of Eca9706 Cell DNA Polβ Gene Targeting Vector

目的构建食管癌细胞Eca9706 DNA聚合酶β(DNA polβ)基因敲除载体,为DNA polβ基因敲除奠定基础。方法应用体细胞基因敲除技术的方法和原理,根据DNA polβ基因序列,设计并合成2对特异性引物(UP1/UP2和DOWN1/DOWN2),通过PCR扩增获得上游同源序列(UP)和下游同源序列(DOWN),其中上游同源序列长1 268 bp,下游同源序列长2 150 bp,将其插入骨架载体pcDNA3.1,从而构建了DNA polβ基因敲除载体pOUT-polβ,最后用PCR、酶切和测序进行鉴定。结果经过PCR筛选,限制性酶切及DNA测序鉴定,证实上游同源序列(UP)和下游同源序列(DOWN)2个片段插入正确。结论通过本研究所述方法,成功构建了用于食管癌细胞Eca9706 DNA polβ基因敲除载体pOUT-polβ。

Objective To construct Eca9706 cell DNA polymerase 13 （DNA polβ） gene targeting vector. Meth- ods According to the principle of somatic gene targeting and DNA polβ sequence, the two special primers were de- signed and synthesized（ UP1/UP2 and DOWN1/DOWN2）to amplify the up homologous sequence and the down ho- mologous sequence. The up sequence（1 268 bp） and the down sequence（2 150 bp） were inserted into skeleton vector pcDNA3.1, then DNA polβ gene targeting vector pOUT-polβ was constructed. Finally, the targeting vector was identified by PCR, restriction enzyme digestion and sequencing. Results The up sequence and the down se- quence were exactly inserted into skeleton vector pcDNA3.1, then DNA poll3 gene targeting vector pOUT-polβ was constructed. Conclusion The Eca9706 cell DNA polβ gene targeting vector was established successfully by using the method in this thesis.