Journal: bioRxiv
Article Title: TET knockout cells transit between pluripotent states and exhibit precocious germline entry
doi: 10.1101/2024.12.02.626356
Figure Lengend Snippet: For all loci the general targeting strategy is indicated (A, C, E) with two gRNAs (scissors) designed close to the start and stop codon, respectively. The position of genotyping primers and the sizes of PCR products are indicated. Open reading frames are indicated in blue and untranslated regions (UTRs) in grey. (B, D, F) agarose gel analysis of PCR genotyping of Tet1, 2 and 3 alleles in all cell lines, with the PCR reaction indicated on the left, and the deduced genotype at the top. A. To knockout Tet1 , wild-type E14Tg2a ESCs were co-transfected with Cas9 and two gRNAs targeting the Tet1 start and stop codons, respectively. B. Two Tet1 -/- clones carrying deletions of both Tet1 alleles (C3 and C5) were obtained, as demonstrated by the presence of a PCR product for the knockout allele (FW1+RV2) and the absence of a PCR product for the wild-type allele (FW1+RV1). C. Tet2 knockout and Tet1, Tet2 double knockout (hereafter referred to as DKO) cell lines were generated from E14Tg2a and Tet1 -/- C5 ESCs using the strategy shown. D. Clones (C26, C34) lacking Tet2 alleles and clones lacking both Tet1 and Tet2 alleles (C13, C19, C24) were generated. E. Tet3 knockout and Tet1, Tet2, Tet 3 triple knockout (hereafter referred to as TKO) ESCs lacking all six Tet alleles were generated from E14Tg2a and Tet1 -/- , Tet2 -/- C13 ESCs as illustrated. F. One clone lacking Tet3 alleles (C3) and two TKO clones lacking all alleles for Tet1, Tet2 and Tet3 (C15, C19) were obtained. WT ctrl. Wild-type E14Tg2a ESCs (parental cell line), H 2 O ctrl. Control sample with no DNA template. Targeting events leading to the generation of single and combined Tet knockout ESC lines are detailed in Figure S1. G. Summary of the Tet knockout ESC lines generated in this study and their interrelationships (see ). Homozygous clone numbers are indicated within brackets. New rounds of targeting are indicated with arrows. H. Tet1 , Tet2 and Tet3 mRNA levels in the indicated ESC lines. mRNA levels were quantified by RT-qPCR, normalised to TBP mRNA levels, and expressed relative to levels in wild-type E14Tg2a ESCs (mRNA/TBP/WT ESC). Error bars: standard deviation in 2 independent replicate experiments.
Article Snippet: Guide RNAs were designed ( http://crispr.mit.edu/ ) and cloned into Cas9/gRNA co-expression plasmids carrying a fluorescent reporter (Addgene PX458 containing EGFP, or a custom-made version containing mCherry).
Techniques: Agarose Gel Electrophoresis, Knock-Out, Transfection, Clone Assay, Double Knockout, Generated, Triple Knockout, Control, Quantitative RT-PCR, Standard Deviation
Addgene inc is a verified supplier 
