Skip to main content
. 2015 Dec 15;44(6):e55. doi: 10.1093/nar/gkv1345

Figure 2.

Figure 2.

attH4X targeting in human embryonic stem cell (hESCs). (A) Schematic diagram of pTZ-attP4X-UN-EF1α-eGFP targeting vector after integration into attH4X. Positions of relevant primers, the Southern probe targeting EGFP and HindIII and XbaI restriction sites are indicated. (B) Western blot showing Integrase expression in hESCs. Lysates from hESCs transfected with plasmids expressing Int-C3CNLS (pCMVssInt-C3C), 6xHIS-tagged Int-C3CNLS (pCMVssInt-C3C-H, pEF-Int-C3C-H, pEFssInt-C3C-H) and untransfected control cells were analyzed by western blotting with an anti-HIS tag antibody (top panel). Purified HIS-tagged Integrase C3 was employed as positive control. β-actin was used as loading control (bottom panel). (C) Example of screening for attH4X × attP4X recombination events in hESCs. PCR was performed with genomic DNA (extracted from neomycin-resistant, EGFP-positive hESC recombinants) and primers cs_attH4X_F2 and attP rev (for the left junction; top left panel) and cs_attH4X_R2 and pr21 (for the right junction; bottom left panel). PCR amplified products of the expected sizes (278 and 439 bp) were detected in clone #24. The right panel shows a PCR analysis to confirm site-specific recombination in clone #24 using different genomic locus-specific primers. PCR-amplified products of the expected sizes (∼1.25 kb with primers attP rev and 24G-F2, and ∼750 bp with primers pr21 and 24G-R1) were obtained and confirmed by sequencing. W, no DNA template control; ES, negative control (genomic DNA from parental hESCs); +, positive control (genomic DNA from HT1080 clone #19); M, 100 bp DNA ladder; M1, 1 kb DNA ladder; 16 to 27, genomic DNA from neomycin resistant hESC clones obtained through co-transfection of pTZ-attP4X-UN-EF1α-eGFP and pEF1α-ssInt-C3CNLS. (D) Southern blot analysis. Genomic DNA purified from three targeted hESC clones and parental hESC cell lines were digested with HindIII or XbaI. A probe complementary to EGFP was employed. Lanes: M1, 1 kb DNA ladder; m, DNA ladder (TeloTAGGG Telomere Length Assay kit, Roche); ES, parental DNA; 3, 24, 59, genomic DNA from targeted hESC clones; pUN4X (107, 108), copies of linearized targeting vector pTZ-attP4X-UN-EF1α-eGFP. White arrow heads indicate fragments of the expected size in the targeted clones.