Journal:

Article Title: The DNA repair genes XPB and XPD defend cells from retroviral infection

doi: 10.1073/pnas.0509828103

Figure Lengend Snippet: Evaluation of transduction efficiency in DNA repair mutant and rescued cell lines. Isogenic mutant and rescued cell lines were transduced with HIV-based and MMLV-based retroviral vectors pseudotyped with VSV-G. The only ORF of the vectors is GFP driven by a CMV promoter leading to expression of GFP after successful integration. The percentage of cells expressing GFP (GFP+) at 48 h was measured by flow cytometry. Each bar represents an individual cell line. (A) From the most NER activity to the least, XPB cell lines are XPB(F99S) fully complemented with the WT XPB allele (XPB-wt), the TTD patient-derived XPB(T119P) mutant, XPB(F99S) complemented with the XPB(T119P) mutant allele (XPB-prt), and the XP/CS patient-derived XPB(F99S) mutant. Paired t test analysis yielded the following two-tailed P values for HIV-GFP infections: XPB-wt and XPB(T119P), P = 0.4; XPB-wt and XPB-prt, P = 0.04; XPB-wt and XPB(F99S), P = 0.01. Analysis of MMLV-GFP infection of XPB-wt and XPB(F99S) yielded P = 0.003. (B) XPD cell lines include two XP patient-derived XPD(R683W) mutant cell lines [XPD(R683W) #1 and XPD(R683W) #2], and each line complemented with the WT XPD allele (XPD-wt #1 and XPD-wt #2). Paired t test analysis yielded the following two-tailed P values: HIV-GFP infections of XPD-wt #1 and XPD(R683W) #1, P = 0.009; XPD-wt #2 and XPD(R683W) #2, P = 0.0002. Analysis of MMLV-GFP infections: XPD-wt #1 and XPD(R683W) #1, P = 0.0006; XPD-wt #2 and XPD(R683W) #2, P = 0.003. (C) XPA cell lines include a patient-derived XPA mutant cell line [XPA(Y116ter)] and complemented with the WT XPA allele (XPA-wt). MSH2 cell lines include WT MEFs (MSH2+/+) and MSH2−/− littermate MEFs. Error bars indicate the standard deviation between duplicate infected wells. An identical trend was observed in at least three separate experiments for all cell lines.

Article Snippet: The three MMLV vector plasmids include the envelope construct encoding VSV-G, the MMLV packaging construct pHIT60, and the genomic pLEGFP-CI expressing GFP from a CMV promoter (Clontech).

Techniques: Transduction, Mutagenesis, Expressing, Flow Cytometry, Activity Assay, Derivative Assay, Two Tailed Test, Infection, Standard Deviation

Journal:

Article Title: The DNA repair genes XPB and XPD defend cells from retroviral infection

doi: 10.1073/pnas.0509828103

Figure Lengend Snippet: Quantitative PCR analysis of HIV cDNA in XPB and XPD cell lines. Isogenic mutant and rescued XPB and XPD cell lines were transduced with an HIV-based retroviral vector pseudotyped with VSV-G. (A and B) Cells were transduced at 20 MOI293T based on 293T titers. Total DNA extracts were purified at 8, 24, 48, and 72 h. Late reverse transcripts and a cellular genomic marker gene were quantified by qPCR, yielding the number of HIV cDNA molecules per cell. (A) Transductions of XPB(F99S) cells and XPB-wt cells are compared. Paired t test analysis yielded the two-tailed P value for 20 MOI293T P = 0.0005. (B) Transductions of XPD(R683W) #1 cells and XPD-wt #1 cells are compared. Paired t test analysis yielded the two-tailed P value for 20 MOI293T, P < 0.0001. The number of 2LTR circles (C) and integrated proviruses (D) per cell were determined by qPCR. Error bars indicate the SD between duplicate infected wells. An identical trend was observed in at least three separate experiments for all cell lines.

Article Snippet: The three MMLV vector plasmids include the envelope construct encoding VSV-G, the MMLV packaging construct pHIT60, and the genomic pLEGFP-CI expressing GFP from a CMV promoter (Clontech).

Techniques: Real-time Polymerase Chain Reaction, Mutagenesis, Transduction, Plasmid Preparation, Purification, Marker, Two Tailed Test, Infection