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A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
Published on: September 2, 2021
[Construction of FasL siRNA expression vector and its expression in lung cancer cell line A549.]
Haidong Wang1, Yaoguang Jiang, Yidan Lin
1Cardiothoracic Surgery, Southwest Hospital, Third Military Medical University,Chongqing 400038,P.R.China.
Background:
Fas/FasL is a member of Tumor Necrosis Factor (TNF) super family, and related to tumor cell apoptosis. It is hypothesis by forward study that activated lymphocytes is more sensitive with Fas/FasL due to up-regulation of FasL expression, so it can be inverse killedly and cleared by tumor cell. The aim of this investigate is to study the fuction of FasL gene and gene therapy of lung cancer by to down-regulationg the FasL gene expression with a siRNA expression plasmid in lung cancer cell A549 as well as its inverse killing effect between activated T lymphocytes and lung cancer cell A549.
Methods:
Potential RNAi oligonucleotides of FasL was designed and synthesized according to appropriate web site. Then a FasL siRNA plasmid was constructed using a pGCsi-U6 vector.The plasmid was sequenced to confirm the inserted sequence. Western blot analysis was used to assess the levels of FasL proteins after the constructed plasmids have been transfected into A549 cells.
Results:
It was confirmed by sequencing that the plasmid was constructed successfully. The result of Western blot clearly showed that FasL siRNA plasmid inhibited FasL expression in A549 cells.
Conclusions:
The construct of FasL siRNA plasmid is successful. FasL protein expression of A549 cell is effectively inhibited by RNAi .
