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The RCAS System
Different Types of RCAS Vectors
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.
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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.
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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
- 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.
- 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.
- 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.
- 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.
- 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.
- RCAS/RCAN vectors work for expression of RNAi but not for antisense RNA (see Bromberg-White et al., J. Virol. 78: 4914-4916, 2004).
- If you
use the vector in vivo, it can infect nondividing cells, but the titer is lower.
- 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.
- 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.)
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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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