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A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells
Published on: December 12, 2017
[Construction and identification of BIRC5 shRNA lentiviral expression vector]
Guo-zhou Rao1, Ang Li, Yong-jin Zhu
1Medicine College of Xi'an Jiaotong University, Xi'an, China.
Summary
Researchers successfully constructed BIRC5 shRNA lentiviral vectors using RNA interference. These vectors target the BIRC5 gene, laying groundwork for cancer gene therapy research.
Area of Science:
- Molecular Biology
- Gene Therapy
- Oncology
Background:
- The BIRC5 gene, also known as Survivin, is frequently overexpressed in various cancers and is associated with tumor progression and resistance to apoptosis.
- RNA interference (RNAi) is a powerful tool for gene silencing, offering potential therapeutic strategies for cancer by targeting specific oncogenes.
- Lentiviral vectors are efficient delivery systems for genetic material, enabling stable gene expression in target cells, making them suitable for gene therapy applications.
Purpose of the Study:
- To design and synthesize complementary DNA sequences for short hairpin RNA (shRNA) targeting the human BIRC5 gene.
- To construct and identify a lentiviral interference vector capable of expressing BIRC5-specific shRNA.
- To establish a foundation for investigating the therapeutic potential of inhibiting BIRC5 expression in cancer cells.
Main Methods:
- Complementary DNA sequences for three pairs of BIRC5 shRNA and one negative control were designed and synthesized using RNA interference techniques.
- The synthesized sequences were annealed to form double-stranded oligos and subcloned into a lentiviral plasmid vector (pMAGic) after digestion with Age I and EcoR I enzymes.
- Positive clones were screened after transformation into DH5α competent cells and subsequently identified via PCR amplification and DNA sequencing.
Main Results:
- Gel electrophoresis confirmed the presence of PCR amplification products, yielding bands of 335 bp for positive clones and 298 bp for negative control clones.
- DNA sequencing results verified that the designed and synthesized sequences were successfully incorporated into the amplified DNA fragments.
- The successful construction of four pairs of BIRC5 shRNA recombinant lentiviral expression vectors was confirmed.
Conclusions:
- The successful construction of BIRC5 shRNA lentiviral expression vectors provides a crucial tool for further research.
- These vectors lay the foundation for exploring the inhibition of BIRC5 by siRNA targeting tumor cell proliferation and inducing apoptosis.
- This work supports the development of gene therapy strategies aimed at targeting the BIRC5 oncogene in cancer treatment.
