Liao, Xin-Hua(NIH/Niddk) wrote:
>Hi, dear all,
>Since I joined Dicty community, I encountered a lot of technique problems or confusions. Some of them may be not a real problem, and the question may be stupid, but they all bother me for a long time. I put them all here, hope you can help me clarify them.

>2.      G418 selection: for overexpression, after transformation for around 5 days under G418 selection, I spread single cell on KA plate, and then pick single colony to culture in 12-well plate. A lot of them can not expand in the well anymore. Again, I suspect some WT cells survive through the drug selection. 20ug/ml of G418 is used. Even for the real colonies growing up, sometimes only a few of them express target protein, others do not express but are G418 resistant. This will make me picking a stable overexpression cell line is as difficult as screening a knock-out strain!

>Thanks,       Xin-Hua Liao

Dear Xin-Hua,

As a reference, I will describe my experiences about G418 selection of overexpressors: both pkaC and cudA constructs DNAs were transformed into a KO mutant by Ca2+ phosphate precipitation method (suggested by A. Kuspa; in DictyBase). Colonies proliferated in modified HL5 medium (9cm Petri dish) containing 5.5 ug G418/ml. I finally isolated 39 colonies, and growing colonies expanded and RNA expression based on both the construct DNAs in the isolates was confirmed by semi quantitative RT-PCR/reverse transcriptase PCR. All of transformants isolated expressed both the genes, although no cells survived without the DNAs (negative control). However at another set of transformation I isolated 15 colonies, 8 of 15(53%) expressed both genes products.
Good luck.

H. Ochiai
Emer. Prof.
Hokkaido University