RCAS Packaging
Recombinant RCAS (Replication-Competent Avian Sarcoma-Leukosis Virus long terminal repeat with a Splice acceptor) retroviral vectors serve as an efficient tool for stable and efficient transgene delivery into avian and TVA-engineered mammalian systems. This system provides a robust and reliable platform for in vitro and in vivo applications in functional studies, developmental biology, drug development, and modeling of oncogenic pathways.
Type of RCAS offered
- RCAS Bryan Polymerase (BP) strain with envelope specificity to subgroup A (TVA receptor); RCASBP(A)
Service Details
Price and turnaround Price Match
| Scale | Application | Titer | Volume | Price (USD) | Turnaround |
|---|---|---|---|---|---|
| Ultra-purified pilot | Cell culture & in vivo | >1.0 x 107 TU/mL | 100 ul (10x10 ul) | $1,999 |
21-33 days
|
| Ultra-purified medium | >1.0 x 108 TU/mL | 100 ul (10x10 ul) | $3,599 | ||
| Ultra-purified large | >1.0 x 108 TU/mL | 1 ml (10x100 ul) | $6,599 |
Helper virus
Successful transduction using RCAS requires target cells to express the avian TVA receptor. If your system is not compatible, TVA-expressing helper virus can be purchased to facilitate this. More information can be found below:
Control virus
The control RCAS is designed to match the biological application of the custom virus and to be used for testing RCAS transduction. For example, if the custom virus overexpresses a gene, then the control virus provided will be EGFP control RCAS (RCAS overexpressing EGFP). Detailed information on the control virus is shown below:
Shipping and storage
Our RCASBP(A) retrovirus is stored in HBSS buffer and is shipped on dry ice. Upon receiving, it should be stored at -80°C for long term (stable for at least 6 months), or -20°C for use within one week. The shelf life for RCAS virus is approximately one year. Please avoid repeated freeze-thaw cycles of RCAS virus, as this can result in a large titer drop.
Technical Information
RCAS production and quality control (QC)
For RCAS packaging, the expression vector carrying the gene of interest (GOI) is transfected into DF-1 chicken cells. After a 3 to 6 day incubation period, the supernatant is collected before cells are passaged and expanded for RCAS amplification. Following 3 to 5 days of amplification, the virus-containing supernatant is collected and combined with the previously collected supernatant. The pooled supernatants are then concentrated by PEG precipitation. Finally, viral particles are further purified by sucrose gradient ultracentrifugation.
For each recombinant RCAS produced by VectorBuilder, quality control includes titer measurement by TCID50, sterility testing for bacteria and fungi, and mycoplasma detection. If the RCAS vector encodes a fluorescent protein, fluorescence expression is confirmed during virus amplification. For RCAS vectors encoding a drug-selection marker, a transduction test with drug selection will be performed. Additionally, for ultra-purified RCAS, we routinely perform endotoxin assays to check the endotoxin level. To include the endotoxin results in your COA, an extra cost is required. Additional QC services can be provided upon request.
Major applications of RCAS vectors
The ability of RCAS vectors to efficiently deliver genes specifically into TVA-expressing cells makes them useful for a variety of applications in both avian and mammalian systems. Below are some of the major research areas where RCAS vectors are currently being used:
Functional genomics
The RCAS system is a valuable tool for investigating gene function, signaling pathways, and genetic interactions. As RCAS enables efficient in ovo gene manipulation in avian models, these vectors have been used to introduce stable gene overexpression, gene knockdown or knockout, and reporter expression constructs into developing tissues. This allows detailed examination of the roles of specific genes in processes such as cell migration, lineage tracking, and tissue morphogenesis.
Targeted RCAS infection in mammalian models enables delivery of genes, mutant variants, or gene-silencing constructs into specific cell populations, providing precise spatial control for studying gene function. This specificity is achieved by restricting expression of the TVA receptor to defined cell types, often through the use of transgenic mice, for instance Cre-lox mice, or tissue-specific promoters, thereby limiting viral infection to selected cell populations. For example, RCAS-CRISPR/Cas models allow the generation of somatic gene knockouts in vivo, enabling functional analysis without the need to generate germline knockout mice. This approach allows rapid investigation of gene function and facilitates mosaic genetic manipulation within tissues, which is particularly useful for studying gene interactions in complex biological systems.
Developmental biology
RCAS vectors are particularly well suited for developmental biology studies in avian models as they enable transgene expression during early developmental stages. Due to their replication-competent nature in permissive avian cells, viral production is amplified through cell-to-cell spread without the need for helper systems. This is especially powerful in avian embryo models, where localized infection allows spatially- and temporally-controlled gene expression for the study of developmental processes in vivo, for instance neural patterning and limb development.
In mammalian models, the RCAS-TVA system has been used to target neural progenitors, liver progenitors, or hematopoietic cells. RCASBP(A) vectors have been used to express signaling modulators in TVA-expressing neural stem cells to study brain development, and time-controlled injections of RCAS have been used to enable stage-specific manipulations in developing tissues. This ultimately allows for effectively tracking cell fate and lineage during developmental studies.
Cancer modeling
The RCAS-TVA system is a powerful platform for modeling cancer due to its ability to deliver genes in a highly cell type-specific manner. By engineering cells or mice to express the TVA receptor (e.g. under tissue-specific promoters), viral infection - and by extension gene expression - is restricted to defined cell populations. This allows controlled modeling of cancer development while minimizing off-target effects.
RCASBP(A) vectors have been widely used to introduce oncogenes, activated signaling proteins, or CRISPR/Cas constructs into TVA-expressing cells to study tumor initiation, progression, and gene interactions involved in tumorigenesis. Such models are highly valuable for improving our understanding of tumor biology and for evaluating potential therapeutic strategies in a controlled experimental setting.
Experimental validation
Our RCAS virus has been fully validated and exhibits high transduction efficiency both in avian- and mammalian-based host systems.
- Avian Gene Expression
- Mammalian Gene Expression
Figure 1. Generation of EGFP-expressing chicken embryo fibroblast cells using the RCAS vector system. (A) RCAS was packaged and used to infect naïve DF-1 cells (MOI = 3). Fluorescence intensity of EGFP was quantified using flow cytometry four days post-infection. (B) Representative fluorescence microscopy images. Exposure: brightfield=10 ms; EGFP=100 ms. Magnification: 100x. (C) DF-1 cells infected with RCAS-EGFP showed a ~95% increase in EGFP expression.
Figure 2. Generation of EGFP-expressing HEK293T-TVA cells using the RCAS vector system. (A) RCAS was packaged and used to transduce wildtype or TVA-expressing HEK293T cells (MOI = 30). Fluorescence intensity of EGFP was quantified using flow cytometry three days post-transduction. (B) Representative fluorescence microscopy images. Exposure: brightfield=10 ms; EGFP=100 ms. Magnification: 100x. (C) HEK293T-TVA cells infected with RCAS-EGFP showed a ~72% increase in EGFP expression.
How to Order
Customer-supplied vectors
If customer-supplied RCASBP(A) plasmids are needed, please send them to us following the Materials Submission Guidelines. Please strictly follow our guidelines to set up shipment to avoid any delay or damage of materials. All customer-supplied materials undergo mandatory QC by VectorBuilder which may incur a $100 surcharge for each item. Please note that production may not be initiated until customer-supplied materials pass QC.
Resources
FAQ
RCAS generally refers to a family of replication-competent retroviral vectors derived from Avian sarcoma-leukosis virus. VectorBuilder offers the RCASBP(A) vector, a derivative based on the Bryan polymerase (BP) strain with envelope specificity to subgroup A (TVA receptor). This derivative is most commonly used due to its low cytotoxicity and high viral titers.
Recombinant RCAS vectors generated by VectorBuilder can typically be handled in Biosafety Level 1 facilities as they contain an avian envelope and will not infect non-TVA expressing mammalian cells. However, biosafety policies can vary considerably from one institution to another. Therefore, it is the responsibility of the researchers to handle all viral vectors following appropriate biosafety guidelines that apply for their institution.
