The average length of guide sequence reads was 19.9 bp. Nevertheless, one-quarter of the unmapped reads could be due to the relatively poor genetic annotation of the chicken genome, which again emphasizes the limitations of bioinformatics approaches for specific species. Among the 5,209,083 sequence reads, 4,052,174 reads (77.8%) were mapped to chicken genes, and most of those sequences were accompanied by PAM ( Fig. Although the Ensembl database includes 15,916 chicken genes, the number of annotated chicken genes appears to be at least 4000 less than those in other established genetic model vertebrates, such as humans, mice, and zebrafish ( 16). For example, approximately 500,000 of the guide sequences were mapped to chromosome 1, suggesting robust generation of guide sequences from various loci in the genome. To characterize the complexity of the gRNA library, the library was deep-sequenced using Illumina MiSeq and analyzed by an RNA sequencing (RNA-seq) protocol using the Ensembl chicken genome database ( 16) as a reference. Relatively large deletions observed on the cDNA sequence indicate that the clones in the library can sometimes cause even large functional deletions in the corresponding transcripts. Most of the indels were focused around the guide sequences. Sequencing analysis of about 30 IgM cDNA-containing plasmid clones for each sorted sIgM (−) population clarified the insertions, deletions, and mutations on the locus ( Fig. Because the Ig heavy chain genomic locus is poorly characterized and only the rearranged VDJ allele is transcribed, its cDNA, rather than its genomic locus, was analyzed by Sanger sequencing. 3, upper panels), and these sIgM (−) populations were further isolated by fluorescence-activated cell sorting (FACS). Cμ guides 1, 2, and 3 generated 5.9, 11.7, and 9.2% sIgM (−) populations 2 weeks after transduction, as estimated by flow cytometry analysis ( Fig. These lentiviral clones were transduced into the AID −/− DT40 cell line, which constitutively expresses cell surface IgM (sIgM) because of the absence of Ig gene conversion ( 12). 2D) were further tested to functionally validate the guide sequences in the library. ![]() Three guide sequences specific to Cμ ( Fig.
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