Adding a polyA tail to a vector?????

Eric Anderson e-anderson at ski.mskcc.org
Wed Jul 9 08:49:53 EST 1997


In article <33C2DD26.6B41 at bms.com>, watson_j at bms.com wrote:

>> 
>> Greg Nattrass writes:
>> 
>> > I need to generate a polyA tail of approx 70 adenosines for a Xenopus
>> > expression vector. Would Terminal deoxynucleotidyl Transferase (TdT) be
>> > the enzyme to use for this application. I have used it to tail cDNA but
>> > am unsure as to how I should use it on plasmid DNA.
>
>_snip_
>> 
>To which Dom Spinella replied:
>> 
>> Well you could use terminal transferase, but my impression is that
>> homopolymer purine tails spontaneously terminate at about 20 residues
>> (this is certainly true for polyG tails, and I believe true of polyA
>> tails as well -- regardless of the dNTP or TdT concentration).  However,
>> it is not clear to me why you would want to do this for your
>> application.  Why not just use an expression vector with a
>> polyadenylation signal, and let the transfected cells do the work for
>> you?
>
>Unfortunately, you don't transfect Xenopus oocytes; you make cRNA and
>microinject it. That's why he wants the poly A tract. I will ask my
>colleague, who often makes cRNA for Xenopus expression, to see what they
>have done.
>
>AJW
>----------------
>John Watson
>Bristol-Myers Squibb Co.
>watson_j at bms.com

with that in mind, why not just construct a combination vector which could
be used to transcribe cRNA (i.e. one of the pSP vectors from Promega or
some other company) with a polyA-tract region from a mammalian expression
vector?  i'm sure there could be some technical problems that i'm missing
here but as long as there are suitable restriction sites (always an issue
if you use Promega vectors IMHO) it seems to be a plausible idea.  of
course, since i don't work with Xenopus at all or mammalian expression
systems very much there could be something i just don't get.

eric

-- 
Eric C. Anderson
Sloan-Kettering Institute for Cancer Research 
Dept. of Cell Biology and Genetics
Lab: (212) 639-2977
Fax: (212) 717-3298
e-anderson at ski.mskcc.org



More information about the Methods mailing list