Pmd2 g constructs

Manufactured by Addgene

The PMD2.G constructs are lentiviral transfer vectors that contain the VSV-G envelope protein gene. These constructs are commonly used to produce pseudotyped lentiviral particles for gene delivery applications.

Automatically generated - may contain errors

Lab products found in correlation

Endofectin lenti transfection reagent, by GeneCopoeia (4 mentions) Pspax2, by Addgene (4 mentions) Mission shrna bacterial stocks for atg5, by Merck Group (2 mentions) Polyjet reagent, by SignaGen (2 mentions) Crispr cas9 plasmid, by Santa Cruz Biotechnology (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Streptomycin sulfate, by Thermo Fisher Scientific (1 mentions) Penicillin g sodium, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

PMD2.G (2420 citations) PMD2.G and psPAX2 constructs (2 citations)

4 protocols using pmd2 g constructs

Generating ATG5 Knockdown Cell Line

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Mission shRNA bacterial stocks for ATG5 and scrambled Control were purchased from Sigma Aldrich. Lentiviruses were produced in HEK 293TN cells co-transfected using Endo F ectinTM Lenti Transfection Reagent (GeneCopoeia, 1001–01) with a packaging mixture of psPAX2 and pMD2.G constructs (Addgene). Media containing the viruses was used to infect MMC cells; puromycin (1 μg/ml) was used as a selection marker to enrich for infected cells.

Aqbi H.F., Tyutyunyk-Massey L., Keim R.C., Butler S.E., Thekkudan T., Joshi S., Smith T.M., Bandyopadhyay D., Idowu M.O., Bear H.D., Payne K.K., Gewirtz D.A, & Manjili M.H. (2018). Autophagy-deficient breast cancer shows early tumor recurrence and escape from dormancy. Oncotarget, 9(31), 22113-22122.

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Generation and Validation of p53 Knockout and shATG5 H460 Cells

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H460 cells were obtained from ATCC® (NCI-H460; Gaithersburg, MD). p53 knockout H460 cells (H460crp53) were generated as described elsewhere (36 ). Briefly, cells were co-transfected (3 × 10⁶ cells in 10-cm dish) with 1 μg CRISPR-Cas9 plasmid targeting the p53 loci and 1 μg of a homology-directed repair plasmid for p53 (both from Santa Cruz Biotechnology® Inc., Santa Cruz, CA). Cells were transfected using PolyJet™ reagent (SignaGen Laboratories, Rockville, MD) following the manufacturer’s guidelines. After 72 h, cells were exposed to 2.5 μg/ml puromycin with daily media exchanges to replenish the selection agent. After all cells transfected with 1 μg of a control CRISPR/Cas9 plasmid (Santa Cruz Biotechnology, Inc.) were killed (~96 h), the cells were allowed to recover and grow as individual colonies, which were then selected and examined for expression of p53 by Western blotting. shATG5 H460 cells were generated as described below. Mission shRNA bacterial stocks for ATG5 were purchased from Sigma-Aldrich (St. Louis, MO). Lentiviruses were produced in HEK 293TN cells co-transfected using EndoFectin™ Lenti Transfection Reagent (GeneCopoeia, Rockville, MD) with a packaging mixture of psPAX2 and pMD2.G constructs (Addgene, Cambridge, MA). Media containing the viruses was used to infect the H460 cells.

Xu J., Patel N.H., Saleh T., Cudjoe EK J.r., Alotaibi M., Wu Y., Lima S., Hawkridge A.M, & Gewirtz D.A. (2018). Differential Radiation Sensitivity in p53 Wild-Type and p53-Deficient Tumor Cells Associated with Senescence but not Apoptosis or (Nonprotective) Autophagy. Radiation research, 190(5), 538-557.

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Lentiviral Knockdown and Labeling of Breast Cancer Cells

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MCF7 cells were generously gifted by Dr. Charles Clevenger, at Virginia Commonwealth University. MCF7 and T47D cells were cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum (Thermo Scientific, SH30066.03), 100 U/mL penicillin G sodium (Invitrogen, 15140–122), and 100 μg/mL streptomycin sulfate (Invitrogen, 15140–122).
The ATG5-knockdown was generated as follows: Mission shRNA bacterial stocks of ATG5 were purchased from Sigma Aldrich. Lentivirus was produced in HEK 293T cells, which were co-transfected using EndoFectinTM Lenti Transfection Reagent (GeneCopoeia, 1001-01) with a packaging mixture of psPAX2 and pMD2.G constructs (Addgene). Media containing the virus was used to infect the MCF7 cells. Puromycin (1 μg/ml) was used as a selection marker to enrich for the infected cells.
Lentiviral packaging of H2B-GFP vector, pLenti0.3UbCGWH2BC1-PatGFP was carried out in 293FT cells. Exponentially growing MCF7 and T47D cells were infected with the H2B-GFP lentivirus in the presence of polybrene (4µg/ml). GFP positive cells were selected using FACS Aria cell sorter.

Finnegan R.M., Elshazly A.M., Patel N.H., Tyutyunyk-Massey L., Tran T.H., Kumarasamy V., Knudsen E.S, & Gewirtz D.A. (2023). The BET inhibitor/degrader ARV-825 prolongs the growth arrest response to Fulvestrant + Palbociclib and suppresses proliferative recovery in ER-positive breast cancer. Frontiers in Oncology, 12, 966441.

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Generation of p53-Knockout and shATG5 NSCLC Cells

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H460 NSCLC cells were obtained from ATCC (NCI-H460). p53 knockout H460
were generated by co-transfecting cells (3×10⁶ in 10 cm dish)
with 1 μg CRISPR-Cas9 plasmids targeting the p53 loci (Santa Cruz
Biotechnologies; cat #sc-416469) and 1 μg a homology directed repair
plasmid (Santa Cruz, cat. #sc-416469-HDR) expressing a puromycin selection
marker. Cells were transfected using PolyJet reagent (Signagen) following the
manufacturer’s guidelines. After 72 hr, cells were exposed to 2.5
μg/mL puromycin with daily media exchanges to replenish the selection
agent. After all cells in control plates were killed (96 h), puromycin was
removed and the cells allowed to recover and grow as individual colonies, which
were then individually selected and examined for expression of p53. For the
generation of shATG5 H460 cells, Mission shRNA bacterial stocks for ATG5 were
purchased from Sigma Aldrich. Lentiviruses were produced in HEK 293TN cells
co-transfected using EndoFectin^™ Lenti Transfection Reagent
(GeneCopoeia, 1001–01) with a packaging mixture of psPAX2 and pMD2.G
constructs (Addgene). The media including viruses were used to infect H460
cells.

Patel N.H., Xu J., Saleh T., Wu Y., Lima S, & Gewirtz D.A. (2020). Influence of nonprotective autophagy and the autophagic switch on sensitivity to cisplatin in non-small cell lung cancer cells.. Biochemical pharmacology, 175, 113896.

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