RCAS基因表达载体

Overview

The RCAS (Replication-Competent Avian Sarcoma-Leukosis Virus long terminal repeat with a Splice acceptor) retroviral vectors are derived from the Rous sarcoma virus (RSV). Wildtype RSV retains all essential viral genes and the oncogene src. In RCAS vectors, the src oncogene is deleted and replaced with a transgene of interest while maintaining the src splice sites, enabling expression of the inserted gene from a spliced viral transcript driven by the viral long terminal repeat (LTR). As a result, RCAS vectors remain replication-competent in appropriate avian cells and can infect mammalian cells engineered to express the appropriate viral receptor.

Our RCAS expression vector, RCASBP(A), is a derivative based on the Bryan Polymerase (BP) strain with envelope specificity to subgroup A (TVA receptor). The RCASBP strain has been shown to have higher efficiency replication in chicken cells compared to RCAS. In this vector, the wildtype RSV pol gene is replaced with the pol gene from the high-titer Bryan strain, resulting in enhanced viral production. Retroviral infection generally requires a specific interaction between the envelope glycoprotein on the viral surface and its corresponding receptor (TVA) on the target cell surface. Among the five primary envelope subgroups of the Avian Sarcoma-Leukosis Virus (ASLV) family, subgroup A is most frequently used due to its low cytotoxicity and high viral titers. Primary chicken embryo fibroblasts, immortalized chicken embryo fibroblasts (DF-1), and quail fibroblast cell lines (QT6 or QT-35) naturally express TVA and can be infected with subgroup A viruses. In contrast, mammalian cells lack endogenous TVA and must be genetically engineered to express this receptor to enable RCASBP(A) infection.

Following receptor-mediated entry, RCASBP(A) undergoes reverse transcription of its RNA genome and stable integration of the resulting proviral transgene DNA into the host cell genome. Due to its replication-competent nature in permissive avian cells, viral production is amplified through cell-to-cell spread without the need for helper systems. This is particularly powerful in avian embryo models, where localized infection enables spatially and temporally controlled gene expression for the in vivo study of developmental processes such as neural patterning and limb development.

In mammalian systems, RCASBP(A) vectors are widely used in combination with TVA-expressing cells or transgenic mouse models to achieve highly targeted gene delivery. Although viral replication is limited in mammalian cells, stable integration allows sustained expression of oncogenes, tumor suppressors, or reporter genes. The RCAS–TVA system has been extensively applied in oncology research to model tumor initiation, progression, and genetic interactions in vivo, including studies of glioma, sarcoma, and epithelial cancers. By restricting infection to defined cell populations, RCASBP(A) enables precise genetic manipulation while minimizing off-target effects.

For further information about this vector system, please refer to the papers below:

Highlights

This vector system is optimized for stable and efficient transgene delivery into appropriate avian and TVA-engineered mammalian systems, providing a robust and reliable system for in vitro and in vivo applications in functional studies, developmental biology, and modeling of oncogenic pathways.

Experimental validation

Our RCAS vector has been validated for specific and efficient gene expression in TVA-expressing cells as shown in Figure 1 below.

Figure 1. Generation of EGFP-expressing HEK293T-TVA cells using the RCAS vector system. (A) An RCAS vector encoding for EGFP was transfected in DF-1 cells. Upon amplification, RCAS virions were collected 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 three days post-transduction. Exposure: brightfield=10 ms; EGFP=100 ms. Magnification: 100x. (C) HEK293T-TVA cells infected with RCAS-EGFP showed a ~72% increase in EGFP expression.

Advantages

Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, retroviral transduction can deliver genes permanently into host cells due to integration of the cargo into the host genome.

High specificity: Our vector encodes for the envelope glycoprotein that specifically infects cells expressing TVA, enabling precise transgene delivery and minimizing off-target effects.

In vitro and in vivo efficacy: Our vectors are designed to efficiently deliver and express transgenes in cultured avian and other TVA-positive cells as well as in chicken embryos and other TVA-positive animal models, making them effective tools for both in vitro and in vivo studies.

Technical simplicity: These vectors can be produced efficiently in standard avian cell lines without the need for helper systems or specialized equipment, which reduces overall cost.

Safety: Unlike wildtype RSV, our vector lacks the src oncogene, thereby significantly reducing oncogenic risk while still retaining essential viral genes required for replication in avian cells.

Disadvantages

Very limited cargo capacity: The RCASBP(A) vector genome is ~10 kb, which leaves only ~2.5 kb to accommodate the user's DNA of interest. If a large ORF exceeds this size limit, viral titer can be severely reduced.

Replication-competent in avian host cells: The RCASBP(A) vector contains all essential viral genes for replication and, upon infection of avian cells, can naturally replicate and spread to neighboring cells. While RCASBP(A) can infect TVA-expressing mammalian cells, replication is inefficient due to multiple host-level restrictions, rendering it effectively non-replicative in standard mammalian systems.

TVA overexpression required for mammalian cell applications: As mammalian cells do not express the RCASBP(A) receptor, TVA expression is necessary to allow viral entry. RCAS vectors can efficiently infect and deliver transgenes to TVA-positive mammalian cells, although replication remains limited due to host-specific restrictions.

Key components of our vector

RSV LTR: Rous sarcoma virus (RSV) long terminal repeat. The LTRs reside on two ends of the viral genome and point in the same direction, with both promoter and polyadenylation function. The LTR upstream of the viral genome acts as a promoter to drive transcription while the downstream LTR acts as a polyadenylation signal to terminate the upstream transcript.

Gag-Pol-Env: Codes for the viral structural proteins (Gag), enzymes (Pol), and envelope glycoprotein (Env) required for viral particle assembly, replication, and TVA receptor tropism, respectively.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here. Its expression is driven by the ubiquitous promoter function in the 5' LTR.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pBR322 ori: pBR322 origin of replication. Allows plasmid replication in E.coli.

Rop: Repressor of primer. Regulates plasmid copy number by controlling replication initiation at the pBR3322 ori.