I want to transfect Huh7 with two different vectors one containing neomycin resistance gene and the other containing puromycing resistance gene.
Does one of them affect the other or is there any common antibiotic that works on both genes for the selection of transfected cells?
No, this will not work. Resistance against neomycin, an aminoglykoside antibiotic, is based on bacterial genes for aminoglykoside phosphotransferases, either originating from transposon Tn5 or sometimes other transposons. Puromycin is chemically different. resistance is conferred by the PAC-gene, encoding a puromycin N-acetyltranferase. This gene comes from bacteria, too, e.g. from Streptomyces alboniger. The following paper is probably helpful for you: "Replication of a novel subgenomic HCV genotype 1a replicon expressing a puromycin resistance gene in Huh-7 cells". Liang, CY et al. VIROLOGY 333, 41-53 (2005).
No, this will not work. Resistance against neomycin, an aminoglykoside antibiotic, is based on bacterial genes for aminoglykoside phosphotransferases, either originating from transposon Tn5 or sometimes other transposons. Puromycin is chemically different. resistance is conferred by the PAC-gene, encoding a puromycin N-acetyltranferase. This gene comes from bacteria, too, e.g. from Streptomyces alboniger. The following paper is probably helpful for you: "Replication of a novel subgenomic HCV genotype 1a replicon expressing a puromycin resistance gene in Huh-7 cells". Liang, CY et al. VIROLOGY 333, 41-53 (2005).
If you're trying to do a dual transfection (e.g. you want to have the cells maintain both plasmids) you would need them to have separate resistance. You'll want to do a kill-curve for both antibiotics separately, to make sure that either drug is capable of killing your cell line. Then after transfection, you can add both drugs to ensure that you're selecting for double transfected cells.
I agree to the above answers I myself have used the combination of the same two antibiotics in the media for the cells containing two different vectors.
It is in your interest that both the antibiotics are different. By this way you know that only cells which are successfully transfected with both the vectors are selected. As mentioned in above replies, the resistance is conferred by different mechanisms, so these 2 antibiotics may not affect each other, and also a single antibiotic will not be able to provide both the resistance. Even if find one, or use two vectors with same antibiotic, it will not solve the purpose. As the cells will survive due to the resistance provided by any one vector. The other vector may or may not be continuously expressed, or one vector may be lost in subsequent passages. Hence, having two different antibiotics will make sure both the vectors are functioning as planned.
Hope this helps.
Thank you for your help I'll do it according to your suggestions but I want to know the optimal concentration of each vector for the transfection I saw that they using between 2-4ug of vector does this will work or not?? and if this is good I'll use HiPerfect transfection buffer 3ul per complex does this will be good in comparison with the vector conc or not???
The amount for plasmids for transfection depends on type of transfection reagent. It is generally recomended by its instruction. For most of protocols, 5-10 ug of each vector is needed..
Marwa, generaly about 10*5 cells is used for transfection. And for this amount of cells 5-10 microgram of plasmids is needed!
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