miRNA-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 mouse, comprising:
(a) introducing a donor mouse ES cell into a host mouse embryo, wherein the donor mouse ES cell comprises at a genomic locus of interest a modified allele, and wherein the donor mouse ES cell comprises (i) a selection cassette that comprises a selectable marker operably linked to a first promoter, wherein the selection cassette is flanked upstream and downstream with a first and a second recombinase recognition site, wherein the first and the second recombinase recognition sites are orientated relative to one another to direct an excision, and wherein the modified allele is not flanked with the first and the second recombinase recognition sites, and, (ii) a second promoter operably linked to a gene encoding a recombinase that recognizes the first and the second recombinase recognition site and wherein the recombinase gene is operably linked with a 3′-UTR that comprises an miRNA recognition site that is recognized by an miRNA that is active in an undifferentiated cell but is not active in a differentiated cell so that the nucleotide sequence flanked by the first and the second recombinase recognition site is excised from a genome of the differentiated cell where the recombinase gene is expressed; (b) implanting the host mouse embryo comprising the donor mouse ES cell of step (a) into a mouse capable of gestating the host 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 mouse that lacks the selectable marker in its genome and comprises the modified allele.