Journal: iScience

Article Title: CHIP-dependent regulation of the actin cytoskeleton is linked to neuronal cell membrane integrity

doi: 10.1016/j.isci.2021.102878

Figure Lengend Snippet:

Article Snippet: LDN , Miltenyi Biotech , 130-103-925.

Techniques: Transduction, Recombinant, Data-independent acquisition, CRISPR, Control, Expressing, Stable Transfection, Clone Assay, Software

Journal:

Article Title: Genomewide production of multipurpose alleles for the functional analysis of the mouse genome

doi: 10.1073/pnas.0502273102

Figure Lengend Snippet: Site-specific recombinase-induced inversions in FlipRosaβgeo-trapped ES cell lines. (A and B) ES cells were infected with FlipRosaβgeo virus and selected in G418. X-Gal-positive sublines (blue) were electroporated with FLPe (A) or Cre (B) expression plasmids and stained with X-Gal after incubating for 10 days. DNA extracted from blue and white sublines was subjected to a multiplex PCR to identify inversions. Primer positions within FlipRosaβgeo are indicated by large arrows; allele-specific amplification products are visualized on ethidium bromide-stained gels to the right. (C) Sublines of the FS4B6 ES cell line harboring Cre- or FLPe-inverted gene trap insertions were electroporated with both FLPe- and Cre-expression plasmids. The amplification products obtained from the progeny lines by allele-specific PCR are visualized on the ethidium bromide-stained gel to the right. t, trapped allele; inv, inverted allele; re-inv, reinverted allele; M, molecular weight marker (1 kb + ladder, Invitrogen). FS4B6 (1anes 1-3), parental FlipRosaβgeo-trapped ES cell line; FS4B6 C14 (lanes 4-6), Cre-inverted subline; and FS4B6 F14 (lanes 7-9), FLPe-inverted subline.

Article Snippet: Gene trap expressing ES cell lines were selected in 130 μg/ml G418 (Invitrogen), manually picked, expanded, and stored frozen in liquid nitrogen.

Techniques: Infection, Expressing, Staining, Multiplex Assay, Amplification, Molecular Weight, Marker

Journal:

Article Title: Genomewide production of multipurpose alleles for the functional analysis of the mouse genome

doi: 10.1073/pnas.0502273102

Figure Lengend Snippet: Conditional mutation induced by a FlipRosaβgeo gene trap insertion in the RBBP7 gene (ENSEMBL ID: ENSMUSG00000031353). The Q017B06 gene trap cell line (t) was transiently transfected with a FLPe expression plasmid, and several sublines with inverted gene trap cassettes were identified by X-Gal staining and allele-specific PCR (inv). Inverted sublines were then electroporated with a Cre-expression plasmid and enriched for reinversions by selecting in G418 (re-inv). (A) X-Gal staining (Upper) and allele-specific PCR amplification products (Lower) from the trapped RBBP7 locus in trapped (t), inverted (inv), and reinverted (re-inv) Q017B06 cell lines. Primers used for the multiplex PCRs were identical to those shown in the diagrams of Fig. 2. (B) RT-PCR for the amplification of RBBP7 wild-type and trapped fusion transcripts expressed in Q017B06 cells before and after exposure to FLPe and Cre recombinases. The positions of the primers used are shown on top, wherein U19 = 5′-GCT CTT GAC TAG CGA GAG AGA AG-3′, B32 = 5′-CAA GGC GAT TAA GTT GGG TAA CG-3′, U34 = 5′-CCA GAA GGA AAG GAT TAT GC-3′, and U35 = 5′-ACA GAG CAA ATG ACC CAA GG-3′. Amplification products are visualized below on ethidium bromide-stained gels. Amplification of the RNA polymerase II (RNA pol II) transcript serves as a positive control. wt, parental ES cells; t, trapped Q017B06 cells; inv, inverted Q017B06 subline; re-inv, reinverted Q017B06 subline; endo, endogenous transcript; fus, fusion transcript. (C) Western blot analysis of the RBBP7 protein expressed in Q017B06 cells. Crude cell lysates from the F1 (wt), Q017B06 (t), inverted Q017B06 (inv), and reinverted Q017B06 (re-inv) ES cells were resolved by SDS/PAGE and analyzed by Western blotting using the anti-RbAp46 antibody. The anti-lamin A antibody served as a loading control.

Article Snippet: Gene trap expressing ES cell lines were selected in 130 μg/ml G418 (Invitrogen), manually picked, expanded, and stored frozen in liquid nitrogen.

Techniques: Mutagenesis, Transfection, Expressing, Plasmid Preparation, Staining, Amplification, Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Positive Control, Western Blot, SDS Page