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Updated: Jul 26, 2026
Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells
Published on: April 26, 2017
[Construction and identification of the eukaryotic expression vector expressing short hairpin RNA targeting human
Hailang Yu1, Jueyu Zhou, Wei Meng
1Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, China.
Objective:
To construct the eukaryotic expression vector of short hairpin RNA (shRNA) targeting human collagen type 1 alpha 1 (COL1A1) and observe its inhibiting effect on the expression of target gene.
Methods:
The complementary oligonucleotide sequences coding shRNA were designed and synthesized according to the sequence of human COL1A1 gene, and cloned into the linearized pSilencer(TM);2.1-U6 neo vector. The recombinant vector was confirmed by enzyme digestion analysis and DNA sequencing, and then the positive clones were transfected to human breast cancer cell MDA-MB-231 by Lipofectamine(TM);2000. The stable cell line was selected by G418. The expression of COL1A1 gene was detected by semi-quantitative RT-PCR and Western blot analysis.
Results:
Double-enzyme digestion and DNA sequencing verified the correct sequences of the recombinant plasmid pshRNA-COL1A1. Compared with the control group, the expression level of COL1A1 mRNA and protein was inhibited markedly by pshRNA-COL1A1-1 or pshRNA-COL1A1-2 transfection, and the inhibitory rates were respectively (44.41±3.90)%, (63.05±3.13)% in RT-PCR and (45.50±2.71)%, (66.98±2.08)% in Western blot analysis.
Conclusion:
Specific shRNA interference plasmid vector targeting COL1A1 gene mRNA was constructed successfully.
