Naslov
The development of a mouse mutant system for studying the role of NKG2D in vivo

Autori
Zafirova, Biljana ; Antulov, Ronald ; 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
Predavanje

Vrsta recenzije
Domaća recenzija

Ključne riječi
NKG2D; NK cells; EGFP; Cre/loxP technology; Cre-ERT2 recombinase; tamoxifen; CD3 epsilon

Sažetak
NKG2D is an activating receptor expressed on NK, NKT and some subpopulations of T cells (i.e. CD8+ TCRab+ activated and memory T cells, TCRgd+ T cells). It belongs to the C-type lectin-like family of receptors and interacts with stress-induced H60, Rae1 and Mult1 ligands that can be expressed on various cell types. The aim of this work is to generate a mouse mutant system to study the role of NKG2D in the development, activation, and homeostasis of the NKG2D expressing cell types in vivo. Therefore, we have been developing two NKG2D mutant mouse strains: NKG2D- EGFP knock out/in and NKG2D conditional knock out. The NKG2D-EGFP mutation will serve to trace the NKG2D expressing cells and to achieve the conventional inactivation of the gene in the same time. In the NKG2D conditional knock out mouse will be possible to inactivate the gene in a conditional manner, using the Cre/loxP system, to study its role in specific cell types (NK, NKT and T cells) and at the defined time. In order to achieve above mentioned mutations we designed two targeting vectors and made NKG2D gene targeting in Bruce 4 (C57BL/6) embryonic stem (ES) cells. Upon the selection and screening procedure we obtained several homologous recombinants from each targeting. The NeoR deleted ES clones where then microinjected and we obtained several chimeric mice of each injected clone. 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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