Promoter-regulated differentiation-dependent self-deleting cassette

Abstract

Targeting constructs and methods of using them are provided for differentiation-dependent modification of nucleic acid sequences in cells and in non-human animals. Targeting constructs comprising a promoter operably linked to a recombinase are provided, wherein the promoter drives transcription of the recombinase in an differentiated cell but not an undifferentiated cell. Promoters include Blimp1, Prm1, Gata6, Gata4, Igf2, Lhx2, Lhx5, and Pax3. Targeting constructs with a cassette flanked on both sides by recombinase sites can be removed using a recombinase gene operably linked to a 3′-UTR that comprises a recognition site for an miRNA that is transcribed in undifferentiated cells but not in differentiated cells. The constructs may be included in targeting vectors, and can be used to automatically modify or excise a selection cassette from an ES cell, a non-human embryo, or a non-human animal.

1 . A targeting construct, comprising from 5′ to 3′:
 (a) a mouse targeting arm; 
 (b) a first recombinase recognition site; 
 (c) a nucleic acid sequence encoding a selectable marker; 
 (d) a Prm1 promoter operably linked to a recombinase gene, 
 (e) a second recombinase recognition site; and, 
 (f) a mouse targeting arm; 
 wherein the recombinase gene encodes a recombinase that recognizes the first and the second recombinase recognition sites which are oriented to direct an excision, and wherein the Prm1 promoter drives expression of the recombinase gene in differentiated cells and does not drive expression of the recombinase gene in undifferentiated cells. 
 
     
     
         2 . The targeting construct of  claim 1 , further comprising a reporter gene between the 5′ mouse targeting arm and the first recombinase recognition site. 
     
     
         3 . The targeting construct of  claim 2 , wherein the reporter gene is selected from the group consisting of luciferase, lacZ, green fluorescent protein (GFP), eGFP, CFP, YFP, eYFP, BFP, eBFP, DsRed, and MmGFP. 
     
     
         4 . The targeting construct of  claim 3 , wherein the reporter gene is lacZ. 
     
     
         5 . The targeting construct of  claim 1 , wherein the first and the second recombinase recognition sites are lox sites, and the recombinase gene encodes a Cre recombinase. 
     
     
         6 . The targeting construct of  claim 5 , wherein the lox sites are selected from the group consisting of loxp, lox511, lox2272, lox66, lox71, loxM2 and lox5171. 
     
     
         7 . The targeting construct of  claim 1 , further comprising a second promoter, wherein the second promoter is operably linked to the nucleic acid sequence encoding the selectable marker. 
     
     
         8 . The targeting construct of  claim 7 , wherein the second promoter is selected from the group consisting of UbC promoter, an hCMV promoter, an mCMV promoter, a CAGGS promoter, an EF1 promoter, a Pgk1 promoter, a beta-actin promoter, and a ROSA26 promoter. 
     
     
         9 . The targeting construct of  claim 7 , wherein the second promoter is a UbC promoter and is operably linked to a neomycin phosphotransferase (neor) gene. 
     
     
         10 . The targeting construct of  claim 1 , wherein the Prm1 promoter comprises SEQ ID NO: 1. 
     
     
         11 . The targeting construct of  claim 1 , wherein the recombinase directs an excision of the selectable marker and the recombinase gene. 
     
     
         12 - 26 . (canceled)