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expression construct question

expression construct question - Protocols and Methods Forum

expression construct question - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 05-25-2006, 01:59 PM
Rebecca Pickin
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Default expression construct question



I am in the process of generating an expression construct which will
express 2 different cDNAs. Would it be better to use a vector which
contains 2 mcs and 2 promoters (in addition to antibiotic selection for E.
coli and mammalian cells) or could I insert the cDNAs tandem using a CMV
promoter and BGH Poly A?

Thank you!
Becky

Rebecca Roche Pickin, PhD

Post Doctoral Associate
Center For Environmental Health Sciences
College of Veterinary Medicine - Basic Sciences
Mail Stop 6100
Mississippi State, MS 39762



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  #2  
Old 05-26-2006, 12:07 AM
Aawara Chowdhury
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Default expression construct question

In <[Only registered users see links. ].net >,
Rebecca Pickin <[Only registered users see links. ]> wrote:


I've used plasmids with two separate expression cassettes. However,
you cannot insert the two cDNAs in tandem and get expression of the
second cDNA.

Instead, use a vector with two MCS separated by an IRES. Clone the first
cDNA into the first MCS (before the IRES), and the second cDNA into the
MCS that is after the IRES.

We got a plasmid like this from the Aiyar lab.

AC
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Old 07-23-2012, 07:11 AM
Pipette Filler
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Default Re: expression construct question

Quote:
Originally Posted by Aawara Chowdhury View Post
In <[Only registered users see links. ].net >,
Rebecca Pickin <[Only registered users see links. ]> wrote:


I've used plasmids with two separate expression cassettes. However,
you cannot insert the two cDNAs in tandem and get expression of the
second cDNA.

Instead, use a vector with two MCS separated by an IRES. Clone the first
cDNA into the first MCS (before the IRES), and the second cDNA into the
MCS that is after the IRES.

We got a plasmid like this from the Aiyar lab.

AC
--
Email: echo 14232209320335931527179462016806497532155427589018 6P | dc

i WAS WONDERING WHETHER IN THIS CASE ORIENTATION OF FRAMES ARE IMPORTANT TO BE SAME OR NOT? FOR EXAMPLE ORF BEFORE ires (+3) AND THE ONE AFTER IRES AGAIN SHOULD BE +3?
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Old 08-16-2012, 04:36 PM
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Default Re: expression construct question

Quote:
Originally Posted by serema2003 View Post
i WAS WONDERING WHETHER IN THIS CASE ORIENTATION OF FRAMES ARE IMPORTANT TO BE SAME OR NOT? FOR EXAMPLE ORF BEFORE ires (+3) AND THE ONE AFTER IRES AGAIN SHOULD BE +3?
Reading frame does not matter with IRES constructs since each coding sequence will have its own start and termination codon. However with the 2A viral cleavage peptide reading frame is critical.
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