食管癌细胞Eca9706 DNA聚合酶β基因敲除载体的构建

摘要

Objective To construct Eca9706 cell DNA polymerase β( DNA polβ)gene targeting vector. Meth-ods According to the principle of somatic gene targeting and DNA polβsequence,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 polβ 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.%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1268 bp，下游同源序列长2150 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。