The RCAS System

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

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 Neo^R 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.

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.

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.