5 research outputs found
Growth curves of CRISPR progeny.
<p>Eight heterozygous IgH KO6B females and eight control females were weighed weekly for 8 weeks.</p
Strategy for CRISPR targeting.
<p>A. Diagram of the chicken IgH locus present in PGC line 472β138 used for CRISPR targeting. The IgH locus contained a previously obtained knockout of the JH gene segment (JH-KO), between the D cluster and the constant regions (only CΞΌ is shown), which was replaced with a selectable marker cassette. gRNAs 1 through 4 were designed to target a region upstream of the single functional VH region (indicated with an arrow), and gRNA5 was designed to target the EGFP gene. B. PGCs of line 472β138 were transiently transfected with a construct containing Cas9 or Cas9/gRNA5, specific for EGFP. After 9 days in culture, the cells were analyzed by flow cytometry for loss of green fluorescence.</p
Cre recombination of CRISPR-targeted loxP site.
<p>A. Diagram of the targeted IgH locus before and after Cre recombination. A forward primer upstream of the CRISPR-targeted loxP site was used with two different reverse primers downstream of the loxP site in the JH-KO cassette. In the non-recombined allele, the forward and reverse primers are separated by about 28kb on the chromosome. After Cre recombination, a single loxP site and the promoterless neo gene remain, and the primers are either 1.6 or 2kb apart, which amplifies readily. B. PCR of recombined cells. Cre +: gDNA template from 1783β9 cells transfected with Cre; Cre -, parental 1783β9 cells; JH-KO, gDNA from a heterozygous JH-KO bird; NTC, no template control.</p
CRISPR-mediated targeting of IgH KO6B in PGCs.
<p>A. Detailed diagram of the IgH locus. The 122 bp sequence between the 5β and 3β homology regions in IgH KO6B, used to design the gRNAs, is shown at top. The locations of gRNAs 1β4 are indicated with blue lines above the sequence and the protospacer adjacent motifs (PAM) indicated with red lines. The repair vector IgH KO6B (below) contains 5β and 3β homology regions (HR) in yellow, a single loxP site (blue arrowhead), and a hygromycin selection cassette (orange). The locations of the primer binding sites for the 5β and 3β targeting assays are shown as black arrows. The downstream selectable markers in the JH-KO consist of floxed EGFP (green box) and puro gene (blue box), and a promoterless neo gene in opposite orientation (pink box). The loxP sites are blue arrowheads. B. The 5β targeting assay performed on independent, non-clonal cell populations obtained from co-transfection of the four different gRNAs into 472β138 cells along with Cas9 and IgH KO6B. For each gRNA transfection, 3 hygromycin-resistant populations were analyzed. The positive control (+) was a DT40 cell line that contained a knockout of the functional V region [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154303#pone.0154303.ref022" target="_blank">22</a>] and the negative control (-) was the parental IgH KO6B plasmid. C. The 5β and 3β targeting assays performed on 9 independent clones obtained with gRNA2 (there were 12 clones, but clones 4, 7 and 12 grew more slowly and were not tested at this time). Variation in band intensity is likely to be from variation in the template gDNA amount, since the number of cells harvested was not normalized. The negative control (-) was genomic DNA from a JH-KO transgenic bird, and the positive control (+) was a pool of cells (G2) from the gRNA2 experiment in B. NT, no template control. D. The same 5β and 3β targeting assays performed on EGFP+ birds obtained from breeding cell line 1783β10 chimeras to wild type.</p
Germline transmission rates of chimeras made with CRISPR-modified PGCs.
<p>Germline transmission rates of chimeras made with CRISPR-modified PGCs.</p