Journal: STAR Protocols

Article Title: CRISPR-Cas9-mediated insertion of a short artificial intron for the generation of conditional alleles in mice

doi: 10.1016/j.xpro.2023.102116

Figure Lengend Snippet: Alternative splicing events produced from the recombined AIv4 allele (A) Scyl1 transcript expression in the cerebella of CMV-Cre+; Scyl1 +/+ and CMV-Cre+; Scyl1 AIv4Δ/AIv4Δ mice. Data is expressed as mean ± SEM. RNA extracts from 3 CMV-Cre+; Scyl1 +/+ and 3 CMV-Cre+; Scyl1 AIv4Δ/AIv4Δ mice were analyzed in triplicates. (B) RT-PCR products generated from cerebellar total RNA extracts of Scyl1 +/+ , Scyl1 +/AIv4 , Scyl1 AIv4/AIv4 , CMV-Cre+; Scyl1 +/+ , CMV-Cre+; Scyl1 +/AIv4Δ , and CMV-Cre+; Scyl1 AIv4Δ/AIv4Δ mice. Amplicons of 665, 638, and 488 bp, corresponding to the mature form of the Scyl1 transcript, as well as shorter transcript variants 1 and 2 were obtained. These shorter transcripts were observed only in CMV-Cre+; Scyl1 +/AIv4Δ and CMV-Cre+; Scyl1 AIv4Δ/AIv4Δ mice. (C) Schematic representation of the five most abundant RNA transcripts observed in CMV-Cre+; Scyl1 AIv4Δ/AIv4Δ mice (transcripts variants labeled 1–5). RT-PCR products from CMV-Cre+; Scyl1 AIv4Δ/AIv4Δ mice were TOPO cloned. Plasmid DNA was recovered from 24 clones and analyzed by Sanger sequencing. The frequency of each transcript is illustrated to the right of the transcript. 18 out of 24 clones were obtained for transcript variant 1; 3 out of 24 clones were obtained for transcript variant 2; and 1 out of 24 clones were obtained for transcript variants 3, 4, and 5. Transcript variant 1 resulted from the splicing of the AIv4 splice donor site into the splice acceptor site of exon 4. Transcript variant 2 resulted from the splicing of the AIv4 splice donor site into a cryptic splice acceptor site within the 3′ half of exon 3. Transcript variant 3 contained intronic sequences from intron 2 and the recombined AIv4 cassette as well as sequences encoding exon 2–5. Transcript variant 4 contained the expected sequence of the recombined AIv4 cassette. Transcript variant 5 resulted from the splicing of a cryptic splice donor site within the 5′ half of exon 3 into the AIv4 splice acceptor site. (D) Ectopic expression of HA tagged versions of wild type, transcript variant 1, 2 and 5 under the control of the CMV promoter. All three isoforms generated from transcript variants 1, 2 and 5 were expressed at a much lower level than their wild-type, full length, counterpart. The images are representative of 3 independent experiments.

Article Snippet: OneTaq One-Step RT-PCR Kit , New England Biolabs , E5315S.

Techniques: Produced, Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Labeling, Clone Assay, Plasmid Preparation, Sequencing, Variant Assay

Journal: STAR Protocols

Article Title: CRISPR-Cas9-mediated insertion of a short artificial intron for the generation of conditional alleles in mice

doi: 10.1016/j.xpro.2023.102116

Figure Lengend Snippet:

Article Snippet: OneTaq One-Step RT-PCR Kit , New England Biolabs , E5315S.

Techniques: Recombinant, Lysis, Protease Inhibitor, Sequencing, Purification, TA Cloning, Subcloning, Bicinchoninic Acid Protein Assay, Western Blot, Expressing, Mutagenesis, Software