Promoter-regulated differentiation-dependent self-deleting cassette

摘要

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.

主权项

1. A method for making a genetically modified F0 mouse, comprising:
(a) introducing a donor mouse ES cell into a host mouse embryo, wherein the donor mouse ES cell is genetically modified to comprise:
(i) a selection cassette that comprises a selectable marker, wherein the selection cassette is flanked upstream and downstream with recombinase recognition sites, and(ii) a recombinase expression cassette that comprises a recombinase gene operably linked to a Blimp1 promoter, wherein the Blimp1 promoter has the sequence comprising SEQ ID NO:2 or SEQ ID NO:3, and wherein the recombinase expression cassette is flanked upstream and downstream with the recombinase recognition sites; (b) implanting the host mouse embryo of step (a) into a mouse capable of gestating the host mouse embryo; (c) maintaining the mouse of step (b) under conditions sufficient for gestation; and, (d) obtaining from the mouse of step (c) a genetically modified F0 mouse that lacks a selection cassette and the recombinase expression cassette in a germ cell and retains a selection cassette and the recombinase expression cassette in a non-germ cell.