title: Enhanced expression of human cDNA by phosphoglycerate kinase promoter-puromycin cassette in the mouse transthyretin locus.
abstract: To produce a humanized mouse, it is critical to obtain a correct expression of a human gene/cDNA after insertion into a mouse locus. We previously generated a targeted allele in which the PGK-neo cassette, flanked by lox71 and loxP, was inserted into the first exon of the mouse endogenous transthyretin (Ttr) gene in ES cells. Using these ES cells, we showed that a human transthyretin (TTR) cDNA with the PGK-puro cassette can be efficiently inserted into this locus by Cre-mediated recombination, and that the human TTR cDNA was expressed in a tissue-specific manner under the control of the mouse endogenous Ttr promoter. To examine whether the PGK-puro cassette or IRES could affect the expression of human TTR cDNA, we generated four mouse lines using Cre and Flp-mediated recombination. The mouse line containing the PGK-puro cassette, but not IRES, exhibited quantitatively and temporally similar expression of human TTR cDNA. Removal of the PGK-puro cassette significantly downregulated the expression of the cDNA. The insertion of IRES sequence upstream of the human TTR cDNA resulted in decreased expression, even in the presence of the PGK-puro cassette. The mouse line containing IRES, but not PGK-puro, showed the lowest level of expression. These results suggest that the PGK-puro cassette is necessary to obtain the enhanced expression of a co-existing human cDNA in the mouse Ttr locus, even though the expression of co-existing cDNA was under the control of the mouse endogenous promoter.
authors.affiliation: Division of Developmental Genetics, Center for Animal Resources and Development, Institute of Resource Development and Analysis, Kumamoto University, 2-2-1 Honjo, Kumamoto, 860-0811, Japan.
authors.forename: Zhenghua
authors.initials: Z
authors.lastname: Li
authors.forename: Gang
authors.initials: G
authors.lastname: Zhao
authors.forename: Jingling
authors.initials: J
authors.lastname: Shen
authors.forename: Kimi
authors.initials: K
authors.lastname: Araki
authors.forename: Kyoko
authors.initials: K
authors.lastname: Haruna
authors.forename: Seiya
authors.initials: S
authors.lastname: Inoue
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Yamamura
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D019426:Integrases / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000378:metabolism; D011228:Prealbumin / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011691:Puromycin / Q000494:pharmacology*; D011995:Recombination, Genetic; D012270:Ribosomes / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D011228:Prealbumin; D011691:Puromycin; D010735:Phosphoglycerate Kinase; C045073:Cre recombinase; D019426:Integrases