从鸡B淋巴DT40细胞系中克隆β-actin启动子,替换pCDNA3.1(+)载体中的SV40启动子,构建β-octin启动子驱动的Neomycin抗性基因表达框.将克隆的slgM λ轻链基因两侧各约2 kb的序列插入到抗性表达框的两侧作为同源臂.PCR、酶切以及测序结果表明成功构建了靶向slgM λ轻链基因的置换型打靶载体pCDNA-act-neo-HR.为建立slgM λ轻链基因敲除的DT40细胞模型,探究slgM λ轻链在IBDV感染DT40细胞过程中的作用奠定了基础.
游雷鸣
,
罗俊
,
王爱萍
,
张改平
,
卜丹
,
郭亚男
,
祈艳华
. DT40细胞slgM λ轻链基因敲除载体的构建[J]. 华北农学报, 2009
, 24(3)
: 1
-6
.
DOI: 10.7668/hbnxb.2009.03.001
β-actin promotor was cloned from chicken B lympha DT40 cells and used to replace the SV40 promotor of pCDNA3. 1( + )vector,so as to construct the Neomycin resistance express cassette driven byβ 2actin promotor. Then about 2 kb DNA sequenceson each side of the locusof sIgMλgene were inserted into the MCSof the constructed cassette,act2 ing as the homologous arm. PCR identification,restriction enzyme digestion and sequence analysis confirmed that the tar2 geting replacement vector pCDNA2act2neO2HR for sIgMλgene was successfully constructed. This laid the foundation for establishing the model cell lack of sIgMλgene,and further investigating the roles of sIgMλlight chain in the infection of IBDV to DT40 cells.
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