Gene targeting and subsequent site-specific transgenesis at the β-actin (ACTB) locus in common marmoset embryonic stem cells.
Nonhuman primate embryonic stem (ES) cells have vast promise for preclinical studies. Genetic modification in nonhuman primate ES cells is an essential technique for maximizing the potential of these cells. The common marmoset (Callithrix jacchus), a nonhuman primate, is expected to be a useful transgenic model for preclinical studies. However, genetic modification in common marmoset ES (cmES) cells has not yet been adequately developed. To establish efficient and stable genetic modifications in cmES cells, we inserted the enhanced green fluorescent protein (EGFP) gene with heterotypic lox sites into the β-actin (ACTB) locus of the cmES cells using gene targeting. The resulting knock-in ES cells expressed EGFP ubiquitously under the control of the endogenous ACTB promoter. Using inserted heterotypic lox sites, we demonstrated Cre recombinase-mediated cassette exchange (RMCE) and successfully established a monomeric red fluorescent protein (mRFP) knock-in cmES cell line. Further, a herpes simplex virus-thymidine kinase (HSV-tk) knock-in cmES cell line was established using RMCE. The growth of tumor cells originating from the cell line was significantly suppressed by the administration of ganciclovir. Therefore, the HSV-tk/ganciclovir system is promising as a safeguard for stem cell therapy. The stable and ubiquitous expression of EGFP before RMCE enables cell fate to be tracked when the cells are transplanted into an animal. Moreover, the creation of a transgene acceptor locus for site-specific transgenesis will be a powerful tool, similar to the ROSA26 locus in mice.
Department of Physiology, School of Medicine, Keio University, Tokyo, Japan.
Seiji
S
Shiozawa
Kenji
K
Kawai
Yohei
Y
Okada
Ikuo
I
Tomioka
Takuji
T
Maeda
Akifumi
A
Kanda
Haruka
H
Shinohara
Hiroshi
H
Suemizu
Hirotaka
H
James Okano
Yusuke
Y
Sotomaru
Erika
E
Sasaki
Hideyuki
H
Okano
D000199:Actins / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002144:Callithrix / Q000235:genetics*; D002454:Cell Differentiation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018920:Coculture Techniques; D053595:Embryonic Stem Cells / Q000378:metabolism* / Q000637:transplantation; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D056426:Genetic Loci; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D059785:Karyotype; D051379:Mice; D016688:Mice, Inbred NOD; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D013724:Teratoma / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control; D014746:Vimentin / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000378:metabolism
D000199:Actins; D019006:Neural Cell Adhesion Molecules; D011993:Recombinant Fusion Proteins; D014746:Vimentin; D000509:alpha-Fetoproteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins