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in-frame cloning

in-frame cloning - Protocols and Methods Forum

in-frame cloning - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 05-25-2007, 05:03 PM
Pragnya Das
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Default in-frame cloning



Hi

can anyone suggest what "in-frame cloning" means giving me a simple example.
I want to subclone an inser in pCDNA3 to RCAS (A) by adding ClaI sites to
either end. Do I have to worry about keeping the insert "in-frame" in the
RCAS (a viral) vector?

Thanks

Pragnya Das
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  #2  
Old 05-25-2007, 08:19 PM
DK
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Default in-frame cloning

In article <[Only registered users see links. ].net> , "Pragnya Das" <[Only registered users see links. ]> wrote:

"To clone in frame" means to clone in a way that puts your insert
into the same translational frame as the start of the ORF controlled
by promoter under which control you put the expression of your
gene.

DK

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  #3  
Old 05-26-2007, 03:26 AM
Aawara Chowdhury
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Default in-frame cloning

In <[Only registered users see links. ].net> ,
Pragnya Das <[Only registered users see links. ]> wrote:


In-frame cloning has no relevance in the context of RCAS. For others
in this group, RCAS is a replication competent avian retroviral vector
that is based on Rous sarcoma virus (RSV).

To construct RCAS, two fundamental changes were made in RSV Schmitt-Rupin A:

First, the pol gene was replaced with that from the Bryan "hi-titer" strain
of RSV.

Second, the src oncogene was deleted and a ClaI site was left in its place,
although the src splice-acceptor was not deleted.

Thus inserts in the ClaI site are expressed from a spliced RNA, that is
transcribed using the 5'LTR.

Unlike MLV, in RSV the splice-donor lies before the "gag" start codon.

Thus, inserts that make use of the src splice acceptor do not have to be
in-frame with the gag gene.

AC
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