Development of a new strategy for inducible and reversible gene knockout.

Abstract

The ability to inactivate gene function has contributed to functional genetics studies. Current

techniques of gene knockout using Cre-loxP recombination and RNAi have achieved varying

degrees of success despite certain limitations. To overcome these limitations, we initiated the

development of a new strategy to achieve inducible and reversible gene knockout. The

strategy requires two components, a tetracycline inducible repressor (TetR-kid) and a

repressor binding sequence (ΔTRE). TetR-kid was obtained by coupling the Tetracycline

repressor protein (TetR) to the Kid1 protein. ΔTRE is a modified tetracycline response

element (TRE). Here, a Rosa26 targeting vector was constructed in efforts of developing this

strategy. The vector was designed to introduce the TetR-kid transgene into the Rosa26

genetic locus to achieve ubiquitous and uniform expression of TetR-kid. The vector was

obtained by modifying a BAC containing the locus. By recombineering, the TetR-kid

transgene along with an ACN cassette for positive selection was introduced into the locus

followed by a second recombineering step to retrieve the modified BAC fragment into a

vector that contains a negative selection marker, a diphtheria toxin fragment a (DTA) gene.

This targeting vector would be used for gene targeting in ES cells in efforts to generate TetRkid

knock-in mouse.