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Pmissiongagpol

Manufactured by Merck Group
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The PMISSIONgagpol is a laboratory equipment product manufactured by the Merck Group. It functions as a tool for performing polymerase chain reaction (PCR) assays, a widely used technique in molecular biology and genetics. The core function of the PMISSIONgagpol is to facilitate the amplification of specific DNA or RNA sequences.

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Lab products found in correlation

Pmission vsv g, by Merck Group (4 mentions) Mission plko 1 puro non mammalian shrna control shc002, by Merck Group (2 mentions) Plko 1, by Merck Group (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Tf 1 and k562 cells, by American Type Culture Collection (2 mentions) Polybrene, by Thermo Fisher Scientific (1 mentions) Nontarget shrna, by Merck Group (1 mentions)

4 protocols using pmissiongagpol

1

Lentiviral shRNA Knockdown of ALKBH5

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Lentivirus for the short hairpin RNA (shRNA)-mediated knockdown of ALKBH5 was generated by co-transfection of HEK293T cells with a pLKO.1-vector or pLKO.1-vector carrying specific shRNA together with the packaging vector pMISSION-GAG-POL and a vesicular stomatitis virus G protein expressing vector pMISSION-VSV-G. pLKO.1 was purchased from Sigma-Aldrich (SHC001). Two days post-transfection, lentivirus-containing supernatants were harvested, centrifuged to remove cellular debris, and filtered with a 0.45-um filter. Lentivirus production efficiency was determined in parallel using a GFP overexpression lentivirus vector. C33A2 cells were inoculated with stocks of recombinant lentiviruses by centrifugation at 2000 g for 2 h at room temperature in the presence of 10 lg/ml polybrene (Fisher Scientific). Empty pLKO.1-vector was used as negative control. Cells were then resuspended and grown in normal RPMI media for 2 days, after which transduced cells were selected in the presence of puromycin (1 ug/ml). Cells were either harvested for Western blotting or for RNA extraction and RT-PCR.
Cui X., Nilsson K., Kajitani N, & Schwartz S. (2022). Overexpression of m6A-factors METTL3, ALKBH5, and YTHDC1 alters HPV16 mRNA splicing. Virus Genes, 58(2), 98-112.
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2

Lentiviral shRNA Knockdown Assay

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Lentiviral shRNA vectors (pLKO.1) were either purchased from Sigma or assembled by cloning the shRNA hairpin loop sequence into the AgeI/EcoRI sites of the empty vector. The target sequence for the ZRSR2 shRNAs sh1 and sh2 are 5’-CAACAGTTCCTAGACTTCTAT-3’ and 5’-AGCAGCCCTTTCTCTGTTTAA-3’, respectively. MISSION pLKO.1-puro Non-Mammalian shRNA Control (SHC002; Sigma) was used as control vector for transduction. To generate lentiviral particles, 293T cells (kindly provided by Dr. Bing Lim, Genome Institute of Singapore, Singapore) were co-transfected with shRNA plasmid and the packaging plasmids, pMISSIONgagpol and pMISSIONvsvg (Sigma) using Lipofectamine 2000 (Invitrogen). Virus containing supernatant was collected after 48 and 72 hours, filtered through 0.45 μm filter and stored in aliquots at −80°C. TF-1 and K562 cells (American Type Culture Collection) were infected with lentivirus for 2 rounds, 24 hours apart, in the presence of 5 μg/ml protamine sulfate. Transduced cells were selected in puromycin to generate stable knockdown cell lines. The knockdown was verified using qPCR and western blotting.
Madan V., Kanojia D., Li J., Okamoto R., Sato-Otsubo A., Kohlmann A., Sanada M., Grossmann V., Sundaresan J., Shiraishi Y., Miyano S., Thol F., Ganser A., Yang H., Haferlach T., Ogawa S, & Koeffler H.P. (2015). Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome. Nature communications, 6, 6042.
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3

Lentivirus Production for shRNA Knockdown

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To obtain lentivirus supernatants, HEK293T cells were transiently transfected with pLKO.1-puro-CMV-tGFP containing shRNA sequences (Sigma-Aldrich Japan), pMISSION GAG POL (Sigma-Aldrich Japan), and pMISSION VSV-G (Sigma-Aldrich Japan). Forty-eight hours later, the viral supernatant was collected and utilized for infection. The vector-transduced cells were selected by medium containing puromycin (1.0 μg/mL for HL60 and 2.5 μg/mL for primary samples) and subjected to assays in vitro. Non-target shRNA was a nonfunctional construct purchased from Sigma-Aldrich Japan. The target sequences, from 5′ to 3′, were GGGCCTGGAGATTTGGAATAG (ADAM8sh_1) and GGAATAGTCAGGACAGGTTCC (ADAM8sh_2).
Miyauchi M., Koya J., Arai S., Yamazaki S., Honda A., Kataoka K., Yoshimi A., Taoka K., Kumano K, & Kurokawa M. (2018). ADAM8 Is an Antigen of Tyrosine Kinase Inhibitor-Resistant Chronic Myeloid Leukemia Cells Identified by Patient-Derived Induced Pluripotent Stem Cells. Stem Cell Reports, 10(3), 1115-1130.
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4

Lentiviral shRNA Knockdown Assay

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Lentiviral shRNA vectors (pLKO.1) were either purchased from Sigma or assembled by cloning the shRNA hairpin loop sequence into the AgeI/EcoRI sites of the empty vector. The target sequence for the ZRSR2 shRNAs sh1 and sh2 are 5’-CAACAGTTCCTAGACTTCTAT-3’ and 5’-AGCAGCCCTTTCTCTGTTTAA-3’, respectively. MISSION pLKO.1-puro Non-Mammalian shRNA Control (SHC002; Sigma) was used as control vector for transduction. To generate lentiviral particles, 293T cells (kindly provided by Dr. Bing Lim, Genome Institute of Singapore, Singapore) were co-transfected with shRNA plasmid and the packaging plasmids, pMISSIONgagpol and pMISSIONvsvg (Sigma) using Lipofectamine 2000 (Invitrogen). Virus containing supernatant was collected after 48 and 72 hours, filtered through 0.45 μm filter and stored in aliquots at −80°C. TF-1 and K562 cells (American Type Culture Collection) were infected with lentivirus for 2 rounds, 24 hours apart, in the presence of 5 μg/ml protamine sulfate. Transduced cells were selected in puromycin to generate stable knockdown cell lines. The knockdown was verified using qPCR and western blotting.
Madan V., Kanojia D., Li J., Okamoto R., Sato-Otsubo A., Kohlmann A., Sanada M., Grossmann V., Sundaresan J., Shiraishi Y., Miyano S., Thol F., Ganser A., Yang H., Haferlach T., Ogawa S, & Koeffler H.P. (2015). Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome. Nature communications, 6, 6042.
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