Exon-humanized mouse
Provided are a donor vector having an exon-humanized gene in which only exon nucleotide sequences of a mouse gene are replaced with human exon nucleotide sequences, an ES cell in which a mouse endogenous gene is replaced with the donor vector, and a mouse crated by using the ES cell.
1 . A donor vector comprising a fragment of a target gene, wherein the fragment of the target gene comprises four exons and three introns, wherein the four exons are human nucleotide sequences of the target gene as set forth in SEO ID NOs: 4, 6, 8, and 10, and the three introns are mouse nucleotide sequences as set forth in SEO ID NOs: 5, 7, 9 wherein the target gene is a transthyretin gene, wherein the four exons and the three introns are arranged in the following order; Exon 1 as set forth in SEQ ID NO: 4, Intron 1 as set forth in SEQ ID NO:5, Exon 2 as set forth in SEQ ID NO:6, Intron 2 as set forth in SEQ ID NO:7, Exon 3 as set forth in SEQ ID NO:8, Intron 3 as set forth in SEQ ID NO:9, and Exon 4 as set forth in SEQ ID NO: 10.
2 . A method for producing an exon-humanized mouse comprising exons in a mouse transthyretin gene replaced with exons in a human transthyretin gene, which comprises:
(a) introducing the donor vector according to claim 1 and a first vector comprising a guide RNA comprising a target sequence complementary to a site immediately upstream of Exon 1 in the transthyretin gene, tracrRNA, and DNA encoding a DNA-cleaving CRISPR-Cas enzyme and a second vector comprising a guide RNA comprising a target sequence complementary to a site immediately downstream of Exon 4 in the transthyretin gene, tracrRNA, and DNA encoding a DNA-cleaving CRISPR-Cas enzyme into mouse embryonic stem(ES) cells; wherein the Cas enzyme cleaves the sites immediately upstream of the Exon 1 and immediately downstream of the Exon 4 and the donor vector replaces the exons in the mouse transthyretin gene with exons in the human transthyretin gene through recombination;
(b) creating chimeric embryos from the ES cells obtained in the step (a) and transplanting the chimeric embryos into foster mothers to thereby create chimeric mice; and
(c) selecting a male mouse and a female mouse from among the chimeric mice obtained in the step (b) and crossing them to produce pups.
3 . The donor vector according to claim 1 , wherein the donor vector, when introduced into mouse ES cells, expresses transthyretin that is encoded by said exons.
4 . The method according to claim 2 , wherein the donor vector, when introduced into said mouse ES cells, expresses said transthyretin that is encoded by said exons.
5 . The donor vector according to claim 1 , wherein the donor vector, when introduced into mouse ES cells, expresses said transthyretin that is encoded by said exons in a normal expression level and a normal tissue-specific expression pattern when expressed in a mouse.
6 . The method according to claim 4 , wherein the donor vector, when introduced into said mouse ES cells, expresses said transthyretin that is encoded by said exons in a normal expression level and a normal tissue-specific expression pattern in the mouse.