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Ligation of two same plasmid encoding two different genes to make co-expression

cloning molecular biology

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#1 Xenotoxic

Xenotoxic

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Posted 16 April 2013 - 08:19 AM

Dear, molecular biologists.

My labmate is trying to make two constructs that contain different selection marker in order to over-express two different genes into eukaryotic cell (ES).

Unfortunately, the vector containing these two genes do not contain selection markers. Therefore, they cannot be used in over-expression experiment.

My professor hence recommended to clone puromycin and neomycin gene into the vector so they can be used in the over-expression system.

However, I suggested to make new construct by first subcloning the gene of interest into neomycin or puromycin resistant gene containing construct then fuse these two new constructs (same back-bone) after digesting with two different restriction enzymes in MCS. This way, I thought that there is no need to clone neomycin and puromycin both into constructs and could save lots of time.

Then my senior scientist said it is impossible due to following reasons.

1. It is unlikely that these two ligation process will occur due to being very large fragment.
2. There might be problem with expressing two genes even though they have different promoter in a single vector.

However, I am not convinced since there won't be any problem logically. Although I realize that it might not be a realistic approach and there won't be any ideal ligase to promote ligation between 5-6K plasmids.

What do molecular biology experts think about my approach??? What could be the biggest problem?

Thanks.





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