|Application:||Gene expression microarray analysis|
|Number of samples:||12|
|Release date:||Jul 31 2012|
|Last update date:||Aug 23 2018|
The following constructs were used: Mus musculus Klf4 full length, (ref|NM_010637.1) cDNA, Mus musculus cyclin-dependent kinase inhibitor 1A (P21) (Cdkn1a), cDNA (ref|NM_007669.3) were fused in frame to the modified oestrogen receptor ERT2 (Feil et al., 1997) and cloned into a retroviral vector (Mieg3) expressing the constructs together with eGFP after an internal ribosomal entry site . (Williams et al., 2000). For Klf4, a mutant comprinsing the zinc- finger domains only AA 393 – 474 (Klf4 dN) as well as a mutant comprising the entire protein except the zinc fingers AA 1-393 (Klf4-dC) were constructed and fused in frame to the modified oestrogen receptor. BM cells from 5-FU treated mice were infected with viral constructs, sorted for GFP expression (day8), plated in IMDM , 10% FCS, 2mM Gln, 1% P/S, supplemented with 50ng/ml rSCF, 100ng/ml TPO and 100ng/ml G-CSF and cultured for 72h (day8-day10). On day10 cells were stimulated with 4-OHT (1uM) for 16h. After that time, total RNA was isolated by Trizol method. Reference List: Feil,R., Wagner,J., Metzger,D., and Chambon,P. (1997). Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun. 237, 752-757. Williams,D.A., Tao,W., Yang,F., Kim,C., Gu,Y., Mansfield,P., Levine,J.E., Petryniak,B., Derrow,C.W., Harris,C., Jia,B., Zheng,Y., Ambruso,D.R., Lowe,J.B., Atkinson,S.J., Dinauer,M.C., and Boxer,L. (2000). Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency. Blood 96, 1646-1654.