TABLE 1. Standard RCAS Vectors

Vector

Subgroups available Viral genes in vector LTR
Purpose
Titer
Comments


RCASBP


A,B,D,E

gag, pol, env

ALV (enh+)

High level expression from LTR via a spliced RNA

>107

Replication-competent; Bryan-RSV pol

RCAS

A,B,D,E gag, pol, env ALV (enh+) High level expression from LTR via a spliced RNA >106 Replication-competent

RCANBP

A,B,D gag, pol, env ALV (enh+) Expression of genes from an internal promoter >107 Replication-competent; Bryan-RSV pol

RCAN

A,B,D gag, pol, env ALV (enh+) Expression of genes from an internal promoter >106 Replication-competent

RCOSBP

A,B,D gag, pol, env RAV-O (enh-) Moderate level expression from LTR via a spliced RNA >105 Replication-competent; Bryan-RSV pol

RCOS

A,B,D gag, pol, env RAV-O (enh-) Low level expression from LTR via a spliced RNA ~105 Replication-competent

RCONBP

A,B,D gag, pol, env RAV-O (enh-) Expression of genes from an internal promoter >105 Replication-competent; Bryan-RSV pol

RCON

A,B,D gag, pol, env RAV-O (enh-) Expression of genes from an internal promoter ~105 Replication-competent


TABLE 2. Other RCAS Vectors

Vector

Comments

RCASBP(A) DeltaR

This version of RCASBP(A) lacks the redundant viral sequences outside the LTR.  It is useful for analyzing mutations in the PBS and the PPT.

BBAN

This is a replication-defective version of RCAS derived from the Bryan high-titer virus.  It contains a complete copy of Gag-Pol but is missing Env, so it can accommodate a larger insert in the ClaI site (~ 4 kb) relative to conventional RCAS vectors.  It can be efficiently complemented by cotransfecting with an ASLV env gene or with VSV-G.

TFA-NEO

This is a replication-defective transfection plasmid designed to accept (and express) ClaI inserts prepared for use in the RCAS vector system. It was generated by removing the viral coding information (the segments between SacI and ClaI) from RCAS and inserting, in place of the coding region, an oligonucleotide containing sites for NsiI, EcoRV, and NdeI (in order from the SacI site). It must be grown on a dam- E. coli strain to use the ClaI site. To facilitate the selection of stable transformants in eukaryotic cells, TFA-NEO also expresses NeoR under the control of the chicken beta-actin promoter.  The version of the promoter included in the plasmid is relatively weak; this favors the selection of cells that have the plasmid inserted in sites favorable for expression.  The two expression cassettes (viral and beta-actin-neo) are separated by a polylinker (NsiI, SfiI, NotI, EagI) that makes it simple to generate a defined linear DNA for transfection of eukaryotic cells.

pGT-GFP

This is a derivative of RCASBP M2C (4070A) with a GFP insert in the opposite orientation to the viral genes. The GFP has a splice acceptor and a poly(A) signal.  GFP expression depends on the appropriate insertion of the provirus into a gene.

RSVPs

There are two related shuttle vectors, RSVP(A)-Z and RSVP(A)-B.  Both vectors are derived from RCASBP(A).  RSVP(A)-Z expresses zeocin resistance (either in avian cells or in E. coli); RSVP(A)-B expresses blasticidin resistance.  Both vectors contain a Lac operator sequence, which makes it easy to recover the viral DNA.

 


TABLE 3. RCAS Vectors for Use in Mammalian Cells

The vectors designed for use in mammalian cells have an envelope gene from a murine retrovirus instead of
an envelope gene from an ASLV. All are replication defective in mammalian cells.

Vector

env gene Properties


RCASBP M2C (4070A)


amphotropic

The env gene
in this vector was derived from an amphotropic virus.  The parental virus grew poorly and was adapted by passage in DF-1 cells.  It has a point mutation, P242I, in gp70.  It grows to high titer but is toxic to DF-1 cells.

RCASBP M2C (797-8)

amphotropic This vector was adapted from RCASBP M2C (4070A) by passage in chick embryos.  It grows to high titer and is much less toxic to DF-1 cells than RCASBP M2C (4070A).  It has a point mutation, I242T, in gp70.

RCASBP(Eco)

ecotropic DF-1 cells cannot be infected by ecotropic viruses.  RCASBP(Eco) is propagated in the DF-1 derivative DFJ8, which expresses the ecotropic receptor.  The ecotropic env gene did not require adaptation and the virus has little, if any, toxicity in DFJ8.


TABLE 4. Adaptor Plasmids

Adaptor

Polylinker Purpose Comments


Cla12


pUC12

To convert a variety of DNA sequences into
ClaI fragments

It does not interfere with transcription or translation in either orientation.

Cla12Nco

pUC12N To provide the insert with a eukaryotic leader sequence and an intitiator ATG The prototype NcoI/ATG adaptor is used to ensure efficient expression of inserts in RCAS/RCOS vectors.

SACla12Nco

pUC12N To provide insert with a eukaryotic leader and initiator ATG and a splice acceptor It is used with the defective vector BBAN to express genes from the LTR promoter.



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