A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins.
A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) kappa locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igkappa region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras.
Kakitani
Makoto
M
Pharmaceutical Research Laboratory, Pharmaceutical Division, Kirin Brewery Co. Ltd. 3 Miyahara-cho, Takasaki-shi, Gunma 370-1295, Japan.
Oshima
Takeshi
T
Horikoshi
Kaori
K
Yoshitome
Tetsuo
T
Ueda
Akiko
A
Kajikawa
Miwa
M
Iba
Yumi
Y
Ozone
Yoshinao
Y
Ijima
Yuki
Y
Yoshino
Tohko
T
Itoh
Mikiko
M
Seki
Sachiko
S
Aoki
Ayako
A
Ishihara
Toshie
T
Shionoya
Michiyo
M
Makino
Utako
U
Kitada
Rina
R
Ohguma
Atsuko
A
Ohta
Takami
T
Yoshida
Yoshimasa
Y
Kudoh
Hiroe
H
Hanaoka
Kazunori
K
Sibuya
Kazunori
K
Ishida
Isao
I
Kakeda
Minoru
M
Yagi
Mikio
M
Yoneya
Takashi
T
Tomizuka
Kazuma
K
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D002460:Cell Line; D002678:Chimera; D002999:Clone Cells; D004622:Embryo, Mammalian / Q000166:cytology; D004921:Erythropoietin / Q000097:blood / Q000235:genetics; D018390:Gene Targeting; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D011995:Recombination, Genetic; D013234:Stem Cells / Q000166:cytology; D013926:Thrombopoietin / Q000097:blood / Q000235:genetics
D011506:Proteins; D004921:Erythropoietin; D013926:Thrombopoietin