The full-length sequence of circKL (Geneseed Biotech Co., Ltd.) was cloned into a pLCDH vector (BioVector NTCC, Inc.), which was subsequently cotransfected with two assistant vectors pMD2.G (cat. no. 12259; BioVector NTCC, Inc.) and psPAX2 (cat. no. 12260; BioVector NTCC, Inc.) into 293T cells to produce a lentivirus. The control group was treated with the lentiviral vector. Each cell line was then transfected with the circKL overexpression lentivirus. Cells were subsequently selected following exposure to puromycin for 7 days, after which the results were validated by reverse transcription-quantitative (RT-q)PCR analysis.
Pmd2 g
Manufactured by BioVector
Sourced in China
The PMD2.G is a precision pipetting device designed for accurate liquid handling. It features a digital display, adjustable volume settings, and ergonomic design for efficient laboratory work.
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2 protocols using pmd2 g
1
Overexpression of circKL in Cell Lines
2
Lentiviral Vector Construction for NIS Expression
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Lv-EF1α-OCT4-IRES-EGFP was kindly provided by the Institute of Molecular Biology, Chinese Academy of Sciences; pcDNA3.1-NIS was obtained from our own library [11 (link)]. The NIS gene was amplified from pcDNA3.1-NIS by PCR using the primers: forward (5’-GCGCGGATCCCGGGTATCGATGGAGGCCGTG-3’) and reverse (5’-CGCGTCTAGATCAGAGGTTTGTAGGTAGTGAGC-3’), digested with XbaI and BamHI, and cloned into the XbaI and BamHI sites of Lv-EF1α-OCT4-IRES-EGFP generating a functional vector featuring NIS under the control of the human elongation factor-1α (EF1α) promoter (the OCT4 transgene of Lv-EF1α-OCT4-IRES-EGFP was replaced with NIS).
HEK293T cell line (Cell Bank of the Chinese Academy of Science, Shanghai, China) was cultured in RPMI-1640 medium supplemented with 10% FBS(Fetal Bovine Serum) and 1% penicillin/streptomycin.
Virus particles were generated by cotransfection of HEK293T cells with Lv-EF1α-NIS-IRES-EGFP and the three packaging plasmids pRsv-REV, pMDIg-pRRE and pMD2G(Biovector Science Lab, Beijing, China). The virus particles were harvested by collecting the cell culture medium at 48 h post-transfection; the supernatants were filtered through 0.45 µm filters, centrifuged at 10,000 g for 15 min and the resulting pellet was resuspended in 100 μl culture medium.
HEK293T cell line (Cell Bank of the Chinese Academy of Science, Shanghai, China) was cultured in RPMI-1640 medium supplemented with 10% FBS(Fetal Bovine Serum) and 1% penicillin/streptomycin.
Virus particles were generated by cotransfection of HEK293T cells with Lv-EF1α-NIS-IRES-EGFP and the three packaging plasmids pRsv-REV, pMDIg-pRRE and pMD2G(Biovector Science Lab, Beijing, China). The virus particles were harvested by collecting the cell culture medium at 48 h post-transfection; the supernatants were filtered through 0.45 µm filters, centrifuged at 10,000 g for 15 min and the resulting pellet was resuspended in 100 μl culture medium.
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