how the gene cloned into a vector can expressed in mammalian cell without the 5& - What is the mechanism of translation initiaition with the HCM promoter? (Jul/24/2006 )
As I know many of you are expert of cloning. I am new. And I do not understand when I desige to put my target gene into a vector, I only need to desigen a forward primer with ATG start condon and reverse primer with stop condon. Why I do not need to consider the 5'UTR of the mRNA? When 5'UTR is not present, how the translation is initiated in the mammalian cells??
Is it because of the promoter is derived form virus that uses different pathway that is 5'UTR independent???
there are types of vectors which u can use according to your purpose. if you want to just clone a region in order to have it in your stocks for further use or for amplification reasons, a cloning vector will fit you. but if you wish to express your cloned fragment, then you need to clone your fragment into an expression vector. when expression vectors considered, you do not need to worry about 5'UTR, polyA or start and stop codons. the expression vector itself contains those. so the only think you should do is to clone your fragment in the same frame as ATG codon in the vector.
tis is what i know. i hope it will help.
Some people suggest having for eg. 5' UTR of the protein as well in the expression cassette for better expression. Our lab has few constructs with 5' UTR for better expression.
We have some constructs with the Lamin 5'UTR upstream the cDNAs because it gives us better expression. But in general we clone without worrying to much about it because expression plasmids have strong promoters and 5'UTRs
It is very important that if your gene has a Kozak sequence in it's 5' UTR, you should keep this sequence when you clone it.
For more information try (I got hese from Google):