4 replies to this topic

[stable genome]

Hi,

I suspect my protein is involved in particular aspect of DNA metabolism.I found by down regulating the activity has been affected.Now to make sure and to characterize which domain is involved,i like to perform gain of function experiment invivo by
down regulating wild type protein and expressing only truncated version.

Now,do any one have an idea how i can perform this experiment.I should down regulate wild type but by the same time i should express truncated protein.

[T C]

Hey,

You need to mutate and then complement. Alternatively, u can just try overexpression, maybe the response is many folds as compared to wild type.

Best,
TC

stable genome, on Jul 13 2009, 05:41 PM, said:

Hi,

I suspect my protein is involved in particular aspect of DNA metabolism.I found by down regulating the activity has been affected.Now to make sure and to characterize which domain is involved,i like to perform gain of function experiment invivo by
down regulating wild type protein and expressing only truncated version.

Now,do any one have an idea how i can perform this experiment.I should down regulate wild type but by the same time i should express truncated protein.

[Functional Screens]

Here's my suggestion,
1. make sure you get good knockdown on the endogenous gene of your interest by one or two shRNAs.
2. generate silent mutations (1-2 codons) in the shRNA targeting site (around 8th to 12th nucleotides of the target sequence), therefore your exogenous copy will not be targeted by the same shRNAs used to knockdown the endogenous one.
3. express the mutated gene to restore whatever you expected to see (assume the level of expression has minor or no effect).
Good Luck.

[Jon Moulton]

Another strategy:
(1) knockdown the synthesis of the protein with a steric-blocking oligo targeting the 5'-UTR of the mRNA;
(2) clone the cDNA for the gene of interest into an expression vector without the original 5'-UTR sequence, transcribe and collect mRNA, and co-transfect with the steric-blocking oligo. Look for rescue of the original activity.

[Functional Screens]

By targeting the 3'-UTR, Jon's strategy has less cloning work involved. It should be simple and more straightforward.