2 genes on one plasmid

brett brett at BORCIM.WUSTL.EDU
Thu Nov 16 17:42:41 EST 1995


>In article <48ffa7$a8h at biovax.biobase.dk>, wind at biobase.dk (Troels Wind) wrote:
>
>> nathan (nathan at xyppp38.bms.com) wrote:
>> : >>> On 14 Nov 1995 18:01:06 -0600, corboy at utdallas.edu said:
>> 
>> 
>> :       Are you sure you need to use two promoters?  I know of at
>> : least two systems where a single promoter will promote expression of
>> : at least two genes (lac promoter in expression of antibody fab
>> : fragments, t7 promoter: will promote expression of genes until t7
>> : terminator is present)
>> 
>> : Someone correct me if I am wrong...
>> 
>> : nathan
>
>I am not sure what system an original post was talking about, but you may
>surely run into a problem with expression of two genes from the single
>promoter in a plant system were bicistronic mRNAs are not transcribed
>efficiently (I can dig out a reference). I am not sure about mammalian
>systems were multicistronic mRNAs are also quite uncommon (if present at
>all). I think, it may have something to do with cap-ribosome subunits
>interaction during translation initiation. This could make translation of
>distal gene problematic if ribosomes tend to fall of mRNA after meeting a
>termination  codon.
>M.A.

Thus, viral IRES elements are used to initiate translation internally. 
I have used at least two vectors with the same design:
      
             __Pr->gene1/IRES(emcv)/gene2(neoR)/pA/intron__
            (______________________________________________)

This seems to work pretty well in finding stable mammalian lines, since
all G418 resistant cells express my gene of interest.



Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain




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