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TVA
is the product of the tv-a
gene ( Bates,
Young, and Varmus, 1993) and
is a receptor for avian leukosis virus, subgroup A (ALV-A).
The receptor has been found to be a membrane-associated protein with
homology to the ligand-binding repeat of the low-density lipoprotein receptor
(LDLR). The introduction of an
avian tv-a gene into cultured mammalian
cells permits infection by ALV-A and expression of genes carried by ALV-A vectors.
Mammalian
cells lack the tva-a gene and are
resistant to infection with ALV-A. Ectopic expression of tv-a
in mammalian cells allows for viral entry and chromosomal integration by ALV-A.
Viral proteins are poorly expressed and new viral particles are not infectious,
preventing cell-to-cell spread. In addition, the lack of viral protein production
decreases the probability of an immune response by the host.
Steve
Hughes and his colleagues have developed a gene transfer system of retroviral
vectors based on the ALV family of retroviruses (Federspiel
et al, 1994).
This TVA technology has been adapted in the Varmus laboratory to transfer
gain-of-function genes into the neonatal mouse brain in order to develop a mouse
model to study the effects of numerous mutations found in human gliomas
(Holland et al, 1997,
Holland et al, 1998).
Due to the poor expression of ALV-A env gene in infected TVA + mammalian cells, the cells remain susceptible to superinfection with ALV-A vectors, allowing for the transfer of multiple genetic lesions and/or histologic markers into a single TVA+ cell (Holland et al, 1997, Holland et al, 1998). This technology provides a cost-effective alternative to conventional mouse models that require extensive breeding of transgenic and knockout animals in order to study synergistic interactions.
Replicating cells are required for successful ALV infection with the TVA system. In studies involving stationary cells, it may be necessary to stimulate cell proliferation or consider alternate methods that make use of viral pseudotypes (Naldini et al., 1996). A RSV pseudotyped lentitiviral system has recently been reported by Lewis et al.
A
review of the multiple vectors that have been developed to express exogenous
genes in ALV-A has been published (Federspiel
& Hughes, 1997). . Among the helper-independent vectors are two groups, RCAS and
RCAN.
RCAS
(Replication-Competent, ALV-LTR, Splice acceptor)
The
RCAS vector series uses viral LTR to drive expression of the gene of interest.
Of this series, the most commonly used vector for the expression of exogenous
genes is RCASBP(A) (RCAS, Bryan-RSV pol, subgroup A).
This vector can accommodate an insert size no larger than 2.5 kb. When the insert size reaches beyond 3.0 kb, the viral titers
from infected avian cells are significantly reduced. Replication-defective vectors have been utilized when expressing
genes larger than 3 kb (Boerkoel
et al, 1993).
RCAN
(Replication-Competent,
ALV-LTR, No splice acceptor)
The
vectors in the RCAN series should be considered if promoters other than the
LTR are necessary to express an exogenous gene.
Alternate promoters may be needed in order to achieve high levels of
protein expression or to target a specific cell lineage.
Chicken embryo fibroblasts and DF1 (Schaefer-Klein et al, 1998; Himly et al, 1998), a chicken fibroblast cell line developed by Doug Foster, support high-titer replication of ALV-A viruses. DF1 cells are preferred due to their longer life span.
Pseudotyped Lentiviral Vectors
This vector system, developed by Lewis et al, allows infection of non-dividing cells. In addition, a much larger insert size can be accommodated. Since an exogenous promoter rather than viral LTR is utilized to drive expression of genes of interest, levels of expression can be enhanced using promoters such as CMV. To download a PDF file, click on Lewis et. al., 2001
The
DF1 cell line, developed by Doug Foster, can be purchased from ATCC but only
through telephone orders. Note
that this cell line cannot be found on the ATTC Website.
RCASBP(A): please contact Dr. Steve Hughes for a draft sequence.
Recent papers on the development of the TVA technology
Pao et al.: Use of the RCAS vector for introducing rtTA
Reviews on the use of the TVA system
Orsulic, 2002, An RCAS-TVA-based approach to designer mouse models. Mamm Genome 13(10):543-7
Federspiel & Hughes (1997). Retroviral gene delivery. Methods Cell Biol, 52, 179-214.
Issues to consider in the use of the TVA system
Please
click her to read infomation on this topic
Practical Protocols in Using the TVA System
2.
Immunohistochemical staining of paraffin and frozen sections (procedure provided
by A. Leavitt)
Rabbit antibody, developed against a synthetic peptide of TVA, is routinely used for immunohistochemical detection of TVA in tissues (Bates, Young, & Varmus, 1993). The antibody is used at a concentration of 1 ug/ml. Vector ABC kits are recommended and standard protocols provided by the manufacturer should be followed. Antigen retrieval is not necessary.
3.
Immunofluorescent staining
4. In situ hybridization: please send me your protocol to be posted here.
5. Genotyping for the tv-a transgene by PCR
Please
send your suggestions and comments to Yi
Li