(NAS Colloquium) Genetic Engineering of Viruses and Viral Vectors (1996) / Chapter Skim
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Epstein-Barr virus vectors for gene delivery to B lymphocytes
Epstein-Barr virus vectors for gene delivery to B lymphocytes
Pages 11334-11340
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From page 11334... ...
, the EBV DNA cis-acting factor responsible for episome persistence was identified by screening for an EBV DNA sequence that would enable a heterologous plasmid with an expression cassette for toxic drug degradation to efficiently transform latently infected cells to toxic drug resistance. Plasmids containing an EBV DNA segment, designated plasmid origin or grip, transformed EBV-infected cells with higher efficiency than plasmids without the EBV DNA.
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From page 11335... ...
Recent strategies for evaluating the effects of site-specific mutations in the viral genome have enabled a focused assessment of the role of specific genes and intragenic elements in primary B-lymphocyte growth transformation and in lytic EBV infection. The favored approach has been to introduce an EBV DNA fragment with a selectable marker into latently infected cells along with an expression vector for the Z immediate early transactivator of lytic EBV replication (1 i.
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From page 11336... ...
LCLs infected with an EBV recombinant with a stop codon inserted at aa 109 in the EBNA3B reading frame arose at the expected frequency indicating from the start that EBNA3B is not critical for growth transformation of primary B lymphocytes in the context of an otherwise normal EBV genome. The LCLs infected with the EBNA3B stop codon recombinant were indistinguishable in their growth from wild-type-recombinant-infected LCLs and were similar in their response to activation of lytic EBV infection.
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From page 11337... ...
However, if the nontransforming phenotype is due to the EBNA3C deletion, Raji cells could be quite useful for recombinant EBV-based genetic analyses of EBNA3C and surrounding genes in much the same way that the P3HR-1 EBV genome has been particularly useful for analyses of the EBNALP and EBNA2 genes. We therefore transfected Raji cells with EBV DNA fragments that included EBNA3C, induced lytic EBV infection, and assayed the resultant virus stocks for the ability to transform primary B lymphocytes.
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From page 11338... ...
The EBNA3C deletion was also analyzed by PCR and these analyses indicated that the LCLs did not have the deletion of EBNA3C that is characteristic of Raji cells. The wild-type fragment of 3.4-kbp fragment characteristic of wild-type DNA would not be amplified under these conditions but the 368-bp deletion DNA fragment was amplified from the parental Raji cell line and not from the LCLs (Fig.
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From page 11339... ...
The data so far simply indicate that the endogenous Raji genome can be transcomplemented by BALF2 and will produce biologically active virus when the SalIC and EcoRIB EBV DNA fragments are transfected into Raji cells; the transforming virus that results has markers from the transfected DNA, including EBNA3C, which is known to be deleted from Raji and to be important for primary B-lymphocyte growth transformation. Thus, the simplest interpretation of the data is that the transfected DNA provides wild-type EBNA3C to the transformation-marker-rescued recombinants.
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From page 11340... ...
11340 Colloquium Paper: Robertson et al. been made in a Raji-EBV-genome-based packaging cell system, further experiments should specifically address these issues.
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