Naslov
Generation of a new mouse mutant strain with inducible and T cell specific control of cre

Autori
Antulov, Ronald ; Zafirova, Biljana ; Polić, Bojan

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Annual meeting of the Croatian Immunological Society, 2005 / Jonjić, Stipan - Rijeka : Hrvatsko imunološko društvo, 2005

Skup
Annual meeting of the Croatian Immunological Society, 2005

Mjesto i datum
Božava, Hrvatska, 29.09.2005. - 02.10.2005

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
Cre/loxP technology; Cre-ERT2 recombinase; tamoxifen; CD3 epsilon

Sažetak
The inducible inactivation of mouse genes by the Cre/loxP system is an elegant method for studying their roles in specific cell type and at the defined time in vivo, thus avoiding any developmental problem that specific mutation may cause in a complex organism. It requires generation of mouse mutants with loxP-flanked gene(s) of interest and inducible expression of Cre recombinase (Cre). Considering our intention to study the role of several genes (i.e. NKG2D, TCRa) in activation and homeostasis of mature T cell populations, we decided to generate a new mouse mutant strain with inducible and T cell specific expression of Cre by gene targeting. The specificity and inducibility of Cre will be ensured by knocking of the CreERT2 cassette into the CD3ε locus. The expression of the CreER fusion protein (Cre and the mutated ligand binding domain (LBD) of the estrogen receptor (ER)) in normal conditions will form an inactive cytosolic complex with hsp90 proteins. In case of administration, tamoxifen (but not natural ER ligands) will induce activity of Cre by binding to the LBD and releasing the fusion protein from the complex, thus allowing its transport to the nucleus and consequent loxP recombination. In order to generate such a mouse mutant we designed an appropriate targeting vector that was electroporated into the Bruce 4 (C57BL/6) embryonic stem (ES) cells. ES clones that underwent positive (G418) and negative (Gancyclovir) selection were screened by Southern blot for homologous recombination. Nine homologous recombinants were identified out of 340 colonies tested. So far, we have microinjected one ES clone and obtained a mouse with a 70% of coat color chimerism. The male chimera has been further mated with C57BL/6 females in order to gain germ line transmission of the desired mutation.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

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