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CDS CLONING QUERY


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#1 zuda

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Posted 27 July 2012 - 08:35 PM

Hello everyone,
One of my seniors asked a question to me , kindly consider telling me your openion. I have cloned CDS of my gene of interest into pCDNA3 vector. The thing is that the region i cloned included almost 98% of CDS region but it does not include ATG codon. I also checked through qrt-pcr, and the construct is working fine. The question is that, is it fine to clone the cds excluding the initiation codon?
Please let me know your openion.

#2 pDNA

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Posted 28 July 2012 - 01:31 AM

did you also check if the protein is expressed? qrt-pcr only gives you info on the transcription of you gene of interest. For translation a RBS and an ATG or another start codon is essential.

There are alternate start condons as well. Eukaryotes use them rarely, prokaryotes more often (e.g. lacI). If you did not include an ATG maybe there is one further downstream ...then you would have a truncated version of your gene.

What we are talking about? ...eukaryotic or prokaryotic systems?

Regards,
p

#3 zuda

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Posted 28 July 2012 - 04:25 AM

thanks for your reply, my gene of interest is NFkb1, i saw that eventhogh i have not included ATG site in my clone but there is another ATG just after primer binding site. I thought that because i am getting very good over-expression at RNA level so i transfected my mammalian cancer cell lines and performed western blotting.Also it is a fact that NF-κB is expressed at endogenous level, but as you mentioned in your comment, that if i have not included all the CDS than the protein will be truncated. In western blot i can clearly see the 2 subunits of NFKB1 [p50 and p105]. The only thing is that the quality of over-expression is not very high, but still after some repeats i got over-expression (20-30%) in contrast with more than 100 folds increment at RNA level.
I am worried if i could proceed to publish it or not, because someone might claim that i have not included initial ATG site, and if someone uses the primers i have used to check the over-expression, than i am not sure, what will be the probability of protein-over-expression. Please advise me.
i seriously hope i could use my results and constructs because i have seen some papers in which the primers they have mentioned (for CDS cloning) did not cover the initial ATG site.
Looking forward for the reply.

#4 pDNA

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Posted 28 July 2012 - 05:12 AM

okay, ...i'm not a specialist on mammalian expression but as far as i know pcDNA3.1 can also be used for expression in E. coli using the T7 promoter. I have checked the plasmid map and it says that it has an ATG initiation codon downstream of the T7 promoter.

Next time i would spend more time on getting to know your expression vector before you start cloning. It is essential to know the precise features that are down and upstream your MCS.

Good luck!

Regards,
p

#5 zuda

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Posted 28 July 2012 - 05:19 AM

Thanks a lot for your comments, i think you saved me, i will check it and hopefully i can convince others. You are absolutely right, i should have spend more time before considering the parts. Anyways, thanks a lot once again for your kind help and support
Regards :)

#6 pDNA

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Posted 28 July 2012 - 05:53 AM

no problem! you are welcome!

All the best!
Regards,
p




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