mcherry expression cassette  (Addgene inc)

Journal: Viruses

Article Title: Schlafen14 Impairs HIV-1 Expression in a Codon Usage-Dependent Manner

doi: 10.3390/v16040502

Figure Lengend Snippet: Effect of SLFN14 on viral replication. ( A ) Role of the RNA Polymerase III-RIG-I-IFN signaling pathway in the SLFN14 activity. HEK293T cells co-transfected with HIVluc expression plasmid and an empty plasmid (control cells) or a plasmid expressing human SLFN14 were treated or not with inhibitors indicated. Luciferase activity measured in these cells was expressed as % of control cells. Data correspond to a triplicate experiment and are representative of three independent experiments. Although not indicated, statistically significant differences ( p ≤ 0.0001) were found between the control and each of the other groups, as calculated with two-way ANOVA and Dunnett post-hoc tests. ( B ) Effect of SLFN14 on HIV-1 replication. ( I ) CD4 and CXCR4 expression. HEK293T cells were co-transfected with plasmids expressing CD4 and a bicistronic plasmid encoding CXCR4 and mCherry, and either, an empty plasmid (control cells) or a human SLFN14 expression plasmid. CD4 was detected by immunostaining and MFI values of CD4 and mCherry (CXCR4) were quantified by flow cytometry. ( II ) These cells were infected with HIV-1 wild-type and viral replication was followed by measuring HIV-1 p24 in the cell supernatant. Data pertain to a triplicate experiment and they are representative of two independent experiments. Statistically significant differences were calculated with one-way ANOVA and Bonferroni post-hoc tests **** p ≤ 0.0001. Figure is created by Valenzuela et al.

Article Snippet: The CXCR4 expression plasmid contains an independent mCherry expression cassette (bicistronic plasmid). pCI Luc contains firefly luciferase cDNA cloned MluI/Xba I in pCI (Promega, Madison, WI, USA). pNLENG1-ES-IRES (a gift of D.N.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Immunostaining, Flow Cytometry, Infection

Journal: Scientific reports

Article Title: Targeting DNA repair pathways with B02 and Nocodazole small molecules to improve CRIS-PITCh mediated cassette integration in CHO-K1 cells.

doi: 10.1038/s41598-023-29863-8

Figure Lengend Snippet: Figure 1. The PITCh-mediated integration of the RMCE landing pad into the S100A locus of CHO-K1. (a) The location of the S100A cluster. The downstream region of the main cluster was targeted by CRISP-PITCh. (b) The sgRNA1 and sgRNA2 target sites. The PAM sequence of each sgRNA was shown in yellow. (c) Schematic representation of CRIS-PITCh donor vector. The homology arms (30 bp) were located on both sides of the landing pad, and the PITCh sgRNAs flanked them. By Cas9-mediated cleavage of the donor, the MMEJ pathway proceeds with targeted integration. Primer position for junction and out-out PCRs is indicated. (d) Agarose gel of 5’/3’ junction PCR results of stable cell pools of sgRNA1 and sgRNA2. The expected band size for 5’ and 3’ junction PCRs is 900 bp and 680 bp, respectively. L referred to the 1 kb DNA ladder. (e) Agarose gel of out-out PCR results of stable cell pools of sgRNA1 and sgRNA2. The locus-specific primers were used to amplify the whole integrated landing pad resulting in PCR product of the targeted locus band (3800 bp). The wild-type CHO band is 900 bp.

Article Snippet: The all-in-one backbone plasmid containing the Cas9 expression cassette and a gRNA scaffold was obtained from Addgene (Plasmid no. 64324).

Techniques: Sequencing, Plasmid Preparation, Agarose Gel Electrophoresis, Stable Transfection