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. Author manuscript; available in PMC: 2015 Aug 21.
Published in final edited form as: Methods Mol Biol. 2015;1332:161–176. doi: 10.1007/978-1-4939-2917-7_12

Figure 2. Construction of VEGFR-1 conditional targeting vector by γ-Red-mediated homologous recombination (recombineering).

Figure 2

A. pL253Flt plasmid, consisting of a pL253 plasmid backbone and a 14.8 kb fragment of the Vegfr-1 gene encompassing exons 2 and 3 (labeled light blue boxes) and associated intronic sequences. Navy blue box, ampicillin resistance gene (amp); yellow box, thymidine kinase negative selection marker (TK, ganciclovir resistance); bright blue bars (a and b) represent the short homology sequences where homologous recombination occurred between FltBac20 and pL253-based retrieval vector, with their external ends (relative to exons) marking the 5’ and 3’ ends of the retrieved fragment. Purple bars represent intronic fragments (c, intron 2; d, intron 3) used as homology arms for recombineering with pL253Flt. B. Recombination cassette containing the following elements in the 5’ to 3’ direction: an intron 2 fragment (purple bar, c ), loxP (triangle), ~150 bp sequence from the end of intron 2, exon 3 (light blue box), ~ 150 bp from the beginning of intron 3, a second loxP site, neomycin resistance cassette (green box) with polyadenylation signal (pA, red box) at its end and flanked by a pair of Frt sites (orange boxes), and an intron 3 fragment (purple, d). Purple bars labeled c and d mediate integration into pL253Flt by homologous recombination. C. The VEGFR-1 conditional targeting vector generated by homologous recombination between pL253Flt (shown in A) and the recombination cassette (shown in B). Not I, site of linearization before electroporation into ES cells.