Figure 3.

Bxb1-mediated insertion of functional cassettes into the Dnmt1 locus. (A) Schematic outline of the strategy and vectors used to create knockout, GFP knockin and cDNA knockin functionalizations of the Dnmt1^attP/attP cell line. cDNAs can be cloned into the attB-GFP-Stop-Poly(A) vector using the 8-cutters AsiSI and NotI. (B) FACS plot depicting the gating and sorting of mESCs to enrich for cells positive for integration of the knockout cassette (2.05% of parent population) based on GFP expression. (C) The Bxb1 surrogate reporter consists of a constitutive CMV promoter followed by an attP site. If Bxb1 and attB donor plasmid containing GFP is present in the cell, recombination of the donor into the reporter leads to expression of GFP. The Bxb1 surrogate reporter can be used to enrich for successful recombination events by FACS. (D) Gel electrophoresis of the multiplex PCR for wt, Dnmt1^attP/attP (attP/attP), Dnmt1^KO/KO (KO/KO), Dnmt1^cDNA/cDNA (cDNA/cDNA) and Dnmt1^GFP/GFP (GFP/GFP) as well as 1:1 mixtures with Dnmt1^attP/attP genomic DNA, to control for amplification biases. Blue arrows indicate expected sizes of the non-recombined (attP) and recombined allele (attL). (E) DNA methylation levels at the major satellite repeats of Dnmt1^KO/KO cells compared to wt and Dnmt1^attP/attP cells. (F) Western blot analysis of DNMT1 expression levels in wt, Dnmt1^attP/attP and Dnmt1^KO/KO cells generated by Bxb1-mediated insertion of a knockout cassette. (G) Western blot analysis of DNMT1 and GFP expression in Dnmt1^attP/attP and homozygous GFP-knockin cells (Dnmt1^GFP/GFP) generated by Bxb1-mediated insertion. (H) Western blot analysis of DNMT1 and GFP expression in Dnmt1^attP/attP and Dnmt1^cDNA/cDNA cells expressing a GFP-Dnmt1 minigene from the endogenous promoter.