Protein expression in the mammalian cells

[Goodie]

# Do I need mRNA destabilization signals for my gene expression? If so where should I add it?

# It says; "cDNA are often obtained by addition of homopolymeric tails into the 5’ non coding region which should be removed.” Which homopolymeric tails are usually used for this purpose?

# Which sequences on the 3’ end that reduce mRNA stability?

# It says that some genes need introns for expression hence I have to add it in my cDNA. How do introns look like and where should I add it?

# Should splicing sequences be already on the plasmid or should I add it on my insert? Should I add it only when I have introns on my insert or should i always have to add it? If so where should I add it on my insert? How do the splicing sequences be like?


Thanks for any inputs.

 

[Ygggdrasil]

In general, for recombinant protein expression in mammalian cells, you would clone only the protein-coding sequence of a gene into a plasmid. You would not clone in any of the 5' or 3' untranslated sequences from the cDNA nor would you include any introns.