FLP engineering - An Overview
FLP engineering - An Overview
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This reporter was focused on the ubiquitously expressed Rosa26 locus in mES cells35. Two correctly targeted clones (#3 and #9) determined by Southern blot Assessment (Supplementary Fig. S1a exhibits clone #three) were subsequently transfected having a mammalian codon-optimized Vika expression plasmid. Vika activity results in the excision from the neo-halt cassette and expression from the lacZ gene through the Rosa26 promoter. Both of those clones confirmed prominent and equivalent recombination efficiencies right after 24 h and forty eight h, demonstrating the Vika/vox process functions effectively to recombine vox web sites placed during the genome of mES cells (Fig. 1a).
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The integration experiments had been done as described higher than and genomic DNA was isolated through the pooled hygromycin resistance colonies. The following PCR Evaluation did not expose any apparent integration of your reporter into these FRT-like sequences Besides integrating into FL-sixty one (knowledge not demonstrated).
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A lot of the Flp variants that bear the typical at the same time the special mutations can be employed as templates to hurry up the evolution by shuffling their genes to generate libraries that may be then screened to determine the variants with the desired focus on specificity.
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