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The RCAS System
Different Types of RCAS Vectors
       Envelope/host range.
       Expression.
       Replication efficiency/level of expression.
       Specialized RCAS derivatives.
Adaptors

Related Expression Systems
Insert Size
What to Avoid in Vector Design
A Little Advice from the People Who Built the Vectors


The RCAS System

The RCAS vectors are a family of retroviral vectors derived from the SR-A strain of Rous sarcoma virus (RSV), a member of the avian sarcoma-leukosis virus (ASLV) family.  In nature, retroviruses can acquire oncogenes from their hosts.  In every case but one, acquisition of the viral oncogene has resulted in the loss of one or more viral genes.  Most retroviruses that have acquired cellular oncogenes are replication defective; RSV is the exception. RSV contains a full complement of viral genes and the oncogene src.  We have taken advantage of this exception to create a family of replication-competent retroviral vectors (see Table 1 and Table 2).  Although the actual construction is more complex, the basic idea is that the RCAS vectors make it possible to substitute other genes/sequences for src.  The name RCAS stands for Replication-Competent ASLV long terminal repeat (LTR) with a Splice acceptor.  The RCAS vectors retain the src splice site and express an inserted gene via a spliced message.  These vectors will replicate in appropriately chosen avian cells (usually chicken or quail, although others can be used). In some cases, appropriately chosen RCAS vectors will infect, but will not replicate, in appropriately chosen mammalian cells (see Table 3).


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Different Types of RCAS Vectors

To make the RCAS vector system more broadly useful, there are vectors available in which several aspects of the basic vector have been changed.  Table 2 describes these vectors.

Envelope/host range.

Retroviral infection requires the specific interaction between the envelope glycoprotein on the surface of the virus and the cognate receptor on the surface of the cell.  The ASLV family of viruses has five primary envelope types: A, B, C, D, E.  These recognize three distinct cellular receptors: A, C, and B/D/E.  A full discussion of the various ASLV envelopes and their receptors is beyond the scope of this site.  For additional information, we recommend the references provided in Chapter 3: Viral Entry and Receptors of Retroviruses (edited by John M. Coffin, Stephen H. Hughes, and Harold E. Varmus, 1997, Cold Spring Harbor Laboratory Press), which is now available as an online publication through the National Library of Medicine website.  If you are undecided about which envelope to choose, A is usually a good choice; this envelope is not toxic to the host cell and viruses with this envelope usually have the highest titer.

Remember:  In order for the virus to be propagated, your cells must have the appropriate receptor and cannot be sequentially infected with two ASLVs expressing the same envelope.  Standard cells, the DF-1 chicken line (ATCC #CRL-12203) and the QT6 quail line (ATCC #CRL-1708 ) can be infected with subgroup A viruses.  If you want to use chicken cells from a local source, make sure they are compatible with the vector you choose and make sure they are not already infected with ASLV.

If A is generally useful, why use other envelopes?  Using RCAS vectors with two different envelopes (A and B, for example) makes it a simple matter to introduce two genes into a single cell (Givol et al., 1994, 1995, 1998).  We have also prepared versions of RCAS vectors that use envelope genes from murine retroviruses.  Although mammalian cells do not have functional receptors for the standard ASLV envelopes on their surface, the amphotropic versions of the RCAS vectors can infect (but will not replicate in) most mammalian cells.  Alternatively, mammalian cells can be modified to express functional receptors for the A and B/D/E envelopes (see the Mouse System).

Expression.

As has already been mentioned, the RCAS vectors produce a spliced message that will lead to the expression of an appropriately inserted gene.  The src-derived splice acceptor site is retained in the RCAS vectors; this means that expression of the spliced message for the inserted gene is driven by the viral LTR.  For those who want to use another means to drive expression, there is the parallel family of RCAN vectors (Replication-Competent, ASLV LTR, No splice acceptor).  The RCAN vectors differ from the corresponding RCAS vectors only in that they lack the src splice acceptor.  RCAN vectors can express an inserted gene from an appropriate internal promoter; internal promoters can retain their tissue specificity when embedded in an RCAN vector (Petropoulos et al., 1992).  RCAN vectors will also express an inserted gene if it is appropriately linked to an internal ribosome entry site (IRES); alternatively, a splice acceptor can be inserted (for example, using the adaptor plasmid SACla12Nco, described in Table 4.  If you would like to express two (small) genes from a single vector, we recommend you use an RCAS (splice) vector and an IRES.  There is also a gene trap RCANBP vector (pGT-GFP), which can be used in either avian or mammalian cells.  This vector has a green fluorescent protein (GFP) insert in the opposite orientation to the viral genes.  GFP is expressed when the gene trap vector is appropriately inserted into a host gene (Zheng and Hughes, 1999; see also Figure 1 and Gene traps and shuttle vectors below).



(click on the image for a larger view)


Figure 1.  DF-1 cells expressing GFP and mouse heat-stable antigen (HSA).  HSA is visualized by Texas Red immunofluorescence.

Replication efficiency/level of expression.

In some cases (for example, for some in vivo applications), it is helpful to be able to regulate the vector replication rate.  It is also useful to be able to control the level of expression from the LTR, since this also determines (for the RCAS vectors) the level of expression of the inserted gene.  There are two elements in the vector that contribute to the level of replication/expression.  The first is the LTR.  ASLV LTRs have strong enhancers; the promoter in the LTR is expressed at a high level in avian cells.  Some mammalian cells express the ASLV LTR at high levels; others do not.  We have some limited information on this issue; however, it is usually best to test the behavior of the ASLV LTR in a specific mammalian cell.  The LTR from the corresponding endogenous avian retrovirus RAV-O appears to lack a strong enhancer; consequently, expression from the RAV-O LTR is much weaker than from the ASLV LTR.  Vectors that contain the RAV-O LTR are RCOS and RCON (Replication-Competent, RAV-O LTR, Splice or No splice acceptor).

In addition to the LTR itself, the choice of the pol region also affects replication and expression in avian cells.  (The pol region does not appear to have a strong effect on the level of expression in mammalian cells, however.)  The original vector (RCAS) was derived in its entirety from the SR-A strain of RSV.  Substituting the pol region from the Bryan high-titer strain of RSV produced a virus that replicated about one log better than RCAS in chicken cells.  This derivative, now widely used, is called RCASBP (Bryan Polymerase).

Remember:  RCAS and RCASBP are not the same vector.  Please try not to confuse the two.  Calling RCASBP RCAS is incorrect.  In addition to RCAS and RCASBP, there is also RCOS and RCOSBP.  In chicken cells, the difference between the level of each pair of these four vectors (in terms of replication and gene expression) is 5- to 10-fold.  In order, from the lowest to the highest in terms of replication/expression, the vectors are RCOS, RCOSBP, RCAS, RCASBP.  This gives the experimentalist about 4 orders of magnitude in terms of replication between the least efficient (RCOS) and the most efficient (RCASBP).  The corresponding vectors that lack the splice acceptor follow the same order, from lowest to the highest in terms of replication/expression:  RCON, RCONBP, RCAN, RCANBP.

Specialized RCAS derivatives.

Replication-defective derivatives.  Although most RCAS vectors are replication competent, there are two replication-defective derivatives:  BBAN and TFA-NEO.  BBAN is based on a defective strain of RSV, the Bryan high-titer virus.  Basically, BBAN 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 the conventional RCAS vectors (see below).  It can be efficiently complemented by cotransfecting with an ASLV env gene or with VSV-G.  We do not have a complementing chicken cell line, and we no longer recommend the QT6 line originally developed for use with BBAN.

TFA-NEO is not, in the strict sense, a viral vector, but it is a transfection plasmid that is designed to accept (and express) the ClaI inserts prepared for use in the RCAS vector system.  TFA-NEO was generated by removing the viral coding information (the segments between SacI and ClaI) from RCAS and inserting, in place of the coding region, a small oligonucleotide that contains sites for NsiI, EcoRV, and NdeI (in order from the SacI site).  Warning:  In TFA-NEO, the ClaI site is subject to dam methylation.  To use the ClaI site, grow the plasmid on a dam- E. coli strain.  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.

Gene traps and shuttle vectors.  We have also prepared two types of specialized vectors:  a GFP-based gene-trap vector (pGT-GFP) and two shuttle vectors (the RSVPs).  pGT-GFP has a GFP gene inserted in the opposite orientation to the viral genes.  The GFP coding region has a splice acceptor at its 5' end. Insertion of the pGT-GFP vector into the introns of expressed genes can generate a fused message that can lead to GFP expression (Zheng and Hughes, 1999).

Both of the RSVP shuttle vectors are based on RCASBP(A).  One, RSVP(A)-Z, expresses zeocin resistance; the other, RSVP(A)-B, expresses blasticidin resistance.  The RSVPs can be propagated either as viruses in avian cells or as plasmids in E. coli (the selections work in both systems).  The RSVPs have, in addition to the selectable markers, a Lac operator sequence.  This makes it easy to use the LacI protein to recover either integrated or unintegrated viral DNA, which greatly simplifies cloning.  Details are provided in Oh et al., 2002.


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Adaptors

The standard RCAS vector can be replicated in E. coli as a plasmid because it carries a pBR-derived replicon with an ampicillin-selectable marker.  The entire plasmid is approximately 12 kb in length and has relatively few unique sites.  All RCAS/RCAN vectors have a unique ClaI site for inserting a foreign gene.  (For those who are interested in the sequence, there is a second ClaI site, but it is methylated in Dam(+) strains of E. coli.)  There are RCAS vectors that have more than one unique site for inserting foreign genes; however, in most cases, genes/sequences are prepared for insertion into the ClaI site of RCAS vectors by using adaptor plasmids or PCR modification.  Adaptor plasmids are small cloning vehicles that have multiple cloning sites flanked by ClaI sites.  Your favorite gene can be converted into a ClaI segment by cloning it into an adaptor plasmid.  The simplest adaptor plasmids are nothing more than a multiple cloning site flanked by ClaI sites and designed not to interfere with transcription or translation in the context of an RCAS vector (for example, Cla12).  Adaptors can also provide a translational start site (Cla12Nco) and, if desired, a splice acceptor (SACla12Nco).  Table 4 describes these adaptors.


Related Expression Systems

There is an E. coli expression system related to the adaptor plasmid Cla12Nco, which has a unique NcoI site (CCATGG).  The ATG of the NcoI site is an efficient translation start site designed to give high-level expression in the context of the RCAS vectors.  pUC12N is a high-copy bacterial plasmid that also has a unique NcoI site.  The ATG in pUC12N is an efficient translation start site in E. coli.  The polylinkers downstream of the NcoI sites are matched in Cla12Nco and pUC12N.  Any insert set up for expression in one of these plasmids is automatically set up for the other system.  The pUC12N expression system can be used to show that a particular insert is properly set up for expression in Cla12Nco; pUC12N can also be used to prepare material for immunization or biochemical analyses.


Insert Size

There are limits to the size of the insert that the RCAS vectors can carry.  The limit is not defined entirely by insert size; some large inserts are tolerated much better than others that are the same length.  There are few, if any, size-related problems with inserts smaller than 2 kb.  Most, but not all, inserts up to 2.5 kb are well tolerated; most inserts larger than 2.5 kb are not.  We have never received definitive reports of inserts larger than 3 kb that could be reliably replicated in an RCAS vector.  If the insert is too large, part or all of the insert will be lost in the initial rounds of viral replication.  A viral stock will be obtained (often with a brief delay); however, the recovered viruses will not contain the intact insert.


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What to Avoid in Vector Design

  1. Don't include a transcription stop site or a polyadenylation site in your insert.  It will interfere with viral replication; attempts to grow vectors with inserts that contain such sequences will lead to deletions in the insert.

  2. Avoid direct repeats in the inserted gene and make sure the insert does not have significant homology with the vector backbone.  The mechanics of reverse transcription will cause deletions between directly repeated sequences anywhere in the viral genome (or insert).  This process is efficient; the obvious way to avoid the problem is to avoid repeated sequences.  The parental RSV has direct repeats flanking src in the RCAS vectors.  The upstream copy of the repeat has been removed.

  3. Use inserts smaller than 2.5 kb (preferably smaller than 2 kb).  Limits to the insert size are discussed above.  Pushing the limits of insert size is usually more trouble than it is worth.

  4. Unless you want to create a cDNA, avoid splice sites in your inserts.  The nature of the retroviral life cycle (alternating RNA and DNA genomes) gives the opportunity for splicing.  While this can be helpful in generating cDNAs from genomic clones, it can also have unintended consequences.  Remember:  Some prokaryotic genes contain sequences that look like splice acceptor/donor sites to eukaryotic splicing machinery.  Check the sequence carefully before you use it.

  5. It is usually quite difficult to propagate RCAS vectors that express inserts that are toxic to the host cell.  If expression of the insert is harmful to the host, variants are rapidly selected that have deleted the insert.  If you want to know if there is a problem with toxicity, put the insert in both an RCAS vector and the corresponding RCAN vector.  If the insert is rapidly lost from the RCAS vector but not the RCAN vector, there is a problem with toxicity.

  6. RCAS/RCAN vectors work for expression of RNAi but not for antisense RNA (see Bromberg-White et al., J. Virol. 78: 4914-4916, 2004).

  7. If you use the vector in vivo, it can infect nondividing cells, but the titer is lower.

  8. Do not pass a viral stock from one cell culture to another unless it is unavoidable.  With repeated passage, the insert will be lost.  It is better to rederive fresh viral stocks by transfection.

  9. Keep in mind that the standard versions of RCAS are terminally redundant for the ends of the viral genome and contain two copies of the primer-binding site (PBS).  (See also the section Plasmid Encoding an RCAS Vector.)


A Little Advice from the People Who Built the Vectors

The RCAS vectors were built to serve the needs of the research community.  This website was created to help anyone who is curious about the system learn what it will (and what it won't) do and to help anyone who wants to use the system get started.  If you have questions or problems and can't figure out the answers from this website and the literature, feel free to contact us.  But, before you call or send e-mail, please look at the information provided on this website (especially the FAQs and literature sections; this will save us both a lot of time.  If you have questions about a specific vector, please make sure you have the name exactly right, as it was when we sent it to you.  Over the years, we have built hundreds of different RCAS vectors, but we cannot answer any specific questions about the vector if we don't know what it is you have.  If you did not receive the vector from us, we cannot help you.  There are a considerable number of derivative RCAS vectors that we did not create.  Some of them are useful; others are not. We have no way of knowing which are which.



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