in-frame cloning

[Aawara Chowdhury]

In <mailman.1251.1180116563.5139.methods from net.bio.net>,
 Pragnya Das <drpragnyadas from gmail.com> wrote:

> 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?

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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