Quick & Easy Conditional Knock Out Kit - FRT

For fast and simple integration of FRT-sites into large vector plasmids at any intended position (for generation of transgenic mouse models).

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Applications

• This kit is designed to integrate FRT sites in a vector plasmid at any intended position without the necessity to use restriction enzymes within 2-4 weeks

Generation of a conditional knockout construct based on FRT sites.

1. Clone containing gene of interest
A gene of interest (orange) was subcloned from e.g. a BAC into a minimal vector by Red/ET recombination using the BAC Subcloning Kit.

2. Red/ET Recombination
To insert a single FRT site an FRT-PGK-gb2-neo-FRT cassette containing 50 bp homology arms added by PCR amplification is introduced at the position of choice by Red/ET recombination with the Quick & Easy Conditional Knockout Kit FRT.

3. Flp-mediated excision
Flp-mediated excision to leave a single FRT site behind. (See also our FRT selection cassettes and Flp expression plasmids & strains).

4. Red/ET Recombinationn
A second FRT-PGK-gb2-neo-FRT cassette containing 50 bp homology arms added by PCR amplification is introduced at the position of choice by Red/ET recombination using the Quick & Easy Conditional Knockout Kit FRT to yield the desired targeting vector.

Description

The included FRT-PGK-gb2-neo-FRT cassette is designed to allow neomycin / kanamycin selection in prokaryotic and eukaryotic cells. It combines a prokaryotic promoter (gb2) for expression of kanamycin resistance in E.coli with a eukaryotic promoter (PGK) for expression of neomycin resistance in mammalian cells. The included pCI-FLPe expression plasmid is coding for an improved FLP recombinase which shows a four to tenfold higher recombinational activity compared to the wildtype FLP enzyme at temperatures between 37°C (optimal growth temperature for E.coli) and 40°C (mouse body temperature). This kit can also be used to work on bacterial artificial chromosomes. High Red/ET efficiency plus convenient removal of the Red/ET Recombination protein expression plasmid pRedET after recombination.

Contents

• Red/ET Recombination protein expression plasmid pRedET. Any E. coli strain can be made Red/ET proficient by transformation with this plasmid
• FRT-PGK-gb2-neo-FRT template to be used for your own experiments
• Expression plasmid for enhanced FLP recombinase pCI-FLPe
• Positive controls to introduce a FRT site in a 18 kb high copy plasmid
• Detailed protocols, descriptions of plasmids, maps and sequences

Sequences

FRT-PGK-gb2-neo-FRT