6 replies to this topic

[nipun]

Hi everyone,

i wish to study shRNA expression in S. pombe.

but all the literature relating shRNA is in mammalian systems and i cudnt find anything other than that.

Can anyone help me out which promoter should i chose for shRNA expression in s pombe system..??

what else should i take care..??

all your suggestions are most welcome.

deep regards

nipun

[nipun]

nipun, on Oct 16 2009, 05:35 AM, said:

Hi everyone,

i wish to study shRNA expression in S. pombe.

but all the literature relating shRNA is in mammalian systems and i cudnt find anything other than that.

Can anyone help me out which promoter should i chose for shRNA expression in s pombe system..??

what else should i take care..??

all your suggestions are most welcome.

deep regards

nipun

no reply..???
No HELP...

[Functional Screens]

Sorry, I might not be able to help.
My question is why expressing "shRNA" in S. pombe? C elegans can use dsRNA.

[Dr Teeth]

Functional Screens, on Oct 20 2009, 11:49 AM, said:

Sorry, I might not be able to help.
My question is why expressing "shRNA" in S. pombe? C elegans can use dsRNA.

I am not a yeast person, but I, too, would add, why do you want to use an shRNA in S. pombe given the availability of knockouts? Do you just want to reduce, but, not knockout, your gene of interest? If this is the case, can you modify a mammalian shRNA vector by swapping the promoters for those appropriate in yeast (ADH?)?

[nipun]

Functional Screens, on Oct 20 2009, 08:49 AM, said:

Sorry, I might not be able to help.
My question is why expressing "shRNA" in S. pombe? C elegans can use dsRNA.

Hi Functional Screen..
Actually I wish to make a new strain of S. pombe deficient in one particular gene and dont wish to knockdown the gene..

regards
nipun

[nipun]

Dr Teeth, on Oct 20 2009, 10:06 AM, said:

Functional Screens, on Oct 20 2009, 11:49 AM, said:

Sorry, I might not be able to help.
My question is why expressing "shRNA" in S. pombe? C elegans can use dsRNA.

I am not a yeast person, but I, too, would add, why do you want to use an shRNA in S. pombe given the availability of knockouts? Do you just want to reduce, but, not knockout, your gene of interest? If this is the case, can you modify a mammalian shRNA vector by swapping the promoters for those appropriate in yeast (ADH?)?

Hi Dr. Teeth
thanks for your suggestion..
Yes I dont wish to completely knock it down.
regards
nipun

[Functional Screens]

nipun, on Oct 20 2009, 10:32 PM, said:

Functional Screens, on Oct 20 2009, 08:49 AM, said:

Sorry, I might not be able to help.
My question is why expressing "shRNA" in S. pombe? C elegans can use dsRNA.

Hi Functional Screen..
Actually I wish to make a new strain of S. pombe deficient in one particular gene and dont wish to knockdown the gene..

regards
nipun

I see. Are you thinking to use a moderate-potent shRNA to semi-knockdown your gene of interest?
I don't remember, there may be a paper talking about RNAi-like mechanism in yeast. However, are you sure there is a mammalian-equivalent RNAi in S. pombe? In fact, I have expressed miRNA to serve as siRNA to knockdown gene, therefore you can use pol II promoter to express miRNA-like siRNA for knockdown IF S. POMBE CAN PROCESS MIRNA.

Why not play with codon usage to introduce some rarly-used codon (by homologous recombination) to reduce the gene expression instead of using RNAi in pombe?

my two cents.