鸡源细胞基因沉默及快速筛选的实用型双标记RNAi载体

利用人H1 RNA启动子、EGFP基因及Neomycin抗性基因,构建用于禽类细胞基因持续沉默和快速筛选的实用型RNAi载体。在将pCDNA3.1(+)载体上的SV40启动子替换为鸡源的β-actin启动子后,装入EGFP基因表达框以及用于驱动外源shRNA转录的人H1 RNA启动子,构建成同时具有EGFP和Neomycin抗性双标记的RNAi载体,并为载体引入独特设计的含媒介序列的多克隆位点以方便外源shRNA编码小片断插入后的快速筛选,载体设计非常实用。插入靶向EGFP和sIgM λ基因的shRNA编码序列后分别瞬时转染DF-1和DT40细胞,结果显示靶基因表达得到了明显抑制。联用EGFP和Neomycin双标记快速筛选sIgM λ轻链基因稳定沉默的DT40细胞克隆的结果也证实,H1启动子转录shRNA的干扰效果是高效的,双标记筛选策略不仅有效而且方便、快捷。

A Dual-marker Vector for Constant RNAi and Rapid Selection of Stable RNAi Clones in Gallus Cells

A practical vector, termed as pAnGH1, designed for constant RNAi and rapid selection of stable RNAi clones in gallus cells was constructed. It replied on the well-know interference of small hairpin RNA (shRNA) to target gene expression, and choosed the EGFP gene as a visual marker, and neomycin resistance gene controlled by the endogenous chicken β-actin promter as a selection marker to faciliate the visual and rapid selection of stable RNAi clones. Also, the specially designed cassete under human H1 RNA promoter contained BglII and HindIII sites that were spaced by an additional 50bp intermediary sequence, which enabled the rapid PCR-scanning of recombinant clones containing the shRNA-coding insert in that the insertion of foreign shRNA-coding fragment resulted in the loss of the priming sites in intermediary sequence. The shRNA-mediated transient interference of EGFP and sIgM λ were performed in the chicken embryo fibroblast cells DF-I and the chicken B-lymphocyte cells DT40 respectively, which exhibited the remarkable inhibition of target expression. In addtiton, the selection of stable sIgMλ-RNAi clones by the dual marker of EGFP and Neomycin was visual and rapid. These results indicated that this vector was high efficient and more convenient in operation.