Applications & Showcases

Red^®/ET^® Recombineering can be applied to numerous DNA engineering tasks. The following example shows the construction of a conditional knockout targeting vector for the murine homeobox protein Hox-A11 using the BAC Subcloning Kit and the Quick & Easy Conditional Knockout Kit (loxP/cre).

1. PCR
Homology regions are added to the linear minimal vector by PCR amplification using vector-specific primers containing 50 bases homologous to Hox-A11 at the 5 end and Phusion High-Fidelity DNA Polymerase.

2. Red/ET Recombineering
Starting from a BAC containing > 100 kb of genomic DNA from mouse chromosome 6 a 15239 bp fragment is subcloned into the minimal vector by Red/ET Recombineering using the BAC Subcloning Kit.

3. Red/ET Recombineering
To insert a single loxP site a floxed PGK-gb2-neo cassette containing 50 bp Hox-A11 homology arms added by PCR amplification using Phusion High-Fidelity DNA Polymerase is introduced at the position of choice by Red/ET Recombineering using the Quick & Easy Conditional Knockout Kit loxP/cre.

4. Excision by Cre
Cre-mediated excision to leave a single loxP site behind. (See also our loxP selection cassettes and Cre expression plasmids & strains).

5. Red/ET Recombineering
A second floxed PGK-gb2-neo cassette containing 50 bp Hox-A11 homology arms added by PCR amplification using Phusion High-Fidelity DNA Polymerase is introduced at the position of choice by Red/ET Recombineering using the Quick & Easy Conditional Knockout Kit (loxP/cre) to yield a targeting vector for ES cell recombination.