| > "Co-housed FVB/NJ mice without CRISPR-mediated correction were used as the functional-deficient control. Briefly, an sgRNAexpressing plasmid had been coinjected, into FVB/NJ zygotes, with the single-stranded oligodeoxynucleotide (ssODN) donor template and Cas9 protein to generate mosaic F0 founders.1" Following to reference 1: > "The sgRNA plasmid was co-injected with the single-stranded oligodeoxynucleotide (ssODN) donor template and the Cas9 protein into FVB/N zygotes to generate eleven F0 founders. [...] Double-strand breaks (DSB) were detected in 7 of 11 mice [...] The target region was sequenced, revealing that F0 3 and 5 incorporated the donor template precisely in 35.7% and 18.8% of somatic cells, respectively (Fig. 1c), while F0 7 and 8 incorporated indels in the integration, corroborating the unexpected results in the RFLP data. [...] A mixture of 3 ng/mL sgRNA plasmid, 3ng/mL of Cas9 protein (NEB Ipswich, MA), and 1mM ssODN (Integrated DNA Technologies, Iowa) was injected into the pronuclei and cytoplasm of FVB/N inbred zygotes. Zygotes that survived injection were transferred into oviducts of 0.5-day post-coitum, pseudopregnant B6xCBA F1 females and carried to term. The resulting gene-corrected mice were backcrossed, initially into the FVB/N background, to determine the germ-line transmission efficiency of the repair."
https://www.ncbi.nlm.nih.gov/pubmed/27203441 This is missing some crucial info isn't it? How many FVB/N zygotes were injected to generate those 11 original mice? |
Generally, reproducibility in biology papers is but a far away dream.