I want to do a transient transfection using COS-7 cells with a plasmid encoding a neomycin resistance, can I use this as a positive transfected cell selection method?
COS-7 cells transfect with plasmid encoding a protein of interest as well as Neomycin resistance
16+ hrs after transfection apply neomycin (0.5mg/ml) and allow 2 days for non-transfected cells to die
48-72hrs post transfection all cells still living should be a sign for positively transfected and can use those cells for further experimentation.
Has anyone had success with this type of approach? Research has been pointing that stable transfection utilizes neomycin not transient.
Read the papers that Chao added, and that should give you more information on the resistance aspect of the transient transfection. As for the transfection process itself, look for relevant transfection reagents (https://altogen.com/product/cos-7-transfection-reagent-kidney-cells-crl-1651/). Cos-7 is a monkey kidney cell line, and there can distinctive qualities making it less receptive to standard reagents.
Hi! COS-7 is very popular cell line for drugs selection. However, Neomycin should not be used for mammalian expression – instead use G418. This can be confusing since the neo/kan gene confers G418 resistance; however, like gentamicin, neomycin is typically used to suppress bacterial growth. Lonza provides a paper that shows "Guideline for Generation of Stable Cell Lines" (http://www.google.co.jp/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0CB4QFjAA&url=http%3A%2F%2Fwww.lonza.com%2Fgo%2Fliterature%2F72&ei=PX34VK-aG4TpmQXijYGgDw&usg=AFQjCNEhNc9_eMNqmlgIexRLN96iXzBJNw&sig2=ALJQ8yhFOmHqeqdG4h6NQQ&bvm=bv.87519884,d.dGY). In this guideline, they show transfection methods, culture condition for drugs selection, protocol for batch culture and limited dilution for drugs selection. Addgene blog also shows detail information for Positive Selection in Mammalian Cells (http://blog.addgene.org/plasmids-101-mammalian-vectors).
Yes, you can select transient transfected cells with neo selection plasmid. You have to keep the cells under neomycin selection medium 24 to 36h after transfection as we need to give enough time for the plasmid to express neo resistance gene.
If you are lucky, you can also get some permanently transferred cells as there are always possibilities for the genomic insertion of these plasmids!
All the best.
I would recommend to do a kill curve to find how sensitive your cells are to the drug, and make sure to allow enought time for you cell to express your gene of interest and the drug resistance gene.
After transfection, I would allow my cell to express for 24-48 hr, then put them under selection pressure for up to 7 days, to select for positive clones.
Please, take into consideration that a DNA vector, a transfection reagent, expression of an antibiotic resistance (trans)gene, expression of a reporter (trans)gene, and selection by acute/chronic antibiotic treatment may evoke cellular responses that affect the biochemical processes under investigation. In the following review, we demonstrate that an assumption that empty vector-transfected cells preserve the cytogenetic and phenotypic characteristics, and represent the adequate control in transfection experiments is not universally valid. We exemplify a number of studies, which reported obvious genomic, transcriptomic and phenotypic changes of tumor cells after transient/stable transfection of an empty vector. Finally, we conceptualize that the diverse experimental manipulations (e.g., transgene overexpression, gene knock out/down, chemical treatments, acute changes in culture conditions, etc.) may act as a system stress, promoting intensive genome-level alterations (chromosomal instability, CIN), epigenetic and phenotypic alterations, which are beyond the function of manipulated genes.
Please see the following manuscript for details:
Stepanenko AA, Heng HH. Transient and stable vector transfection: Pitfalls, off-target effects, artifacts. Mutat Res. 2017 Jul;773:91-103. doi: 10.1016/j.mrrev.2017.05.002.
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