Biology Reference
In-Depth Information
16
Retroviral Delivery of ECM Genes to Cells
Kyonggeun Yoon and Vitali Alexeev
1. Introduction
The introduction of recombinant DNA has become a common tool for study-
ing functional and structural properties of a wide variety of proteins. Func-
tional analysis of protein can be studied by suppression of gene expression,
thus introducing a plasmid which expresses an antisense RNA in mammalian
cells. Several extracellular matrix proteins require an assembly of subunits to
form functional heteromultimers with distinct features. Thus, expression of a
specific mutant protein can interfere with the assembly of multimeric protein,
resulting in a dominant-negative phenotype. In these studies, an efficient
delivery of DNA into appropriate target cells represents a critical step.
Although many procedures of transfection of the plasmid DNA into mamma-
lian cells are available, viral infection represents a far superior mode of deliv-
ery because retroviral vectors were shown to transduce genes of interest into
tissue-culture cells with success rates approaching 100%. The retrovirus inserts
the viral genome into the chromosome of the infected cell permanently, usu-
ally without any measurable effect on the viability of the infected cells. The
result is an efficient gene-transfer system in which most recipient cells will
incorporate and express the transduced gene.
The retrovirus genome consists of an RNA molecule of 8500 nucleotides
packaged into each viral particle
(1)
. The retrovirus enters the cells via interac-
tion between the viral envelope protein and the appropriate viral receptor pro-
tein on the target cell. Once inside the cell, the enzyme reverse transcriptase
brought in with the capsid makes a DNA copy of the viral RNA molecule to
form a DNA-RNA hybrid duplex. After degradation of RNA, the reverse tran-
scriptase completes a second DNA strand synthesis, generating a double-
stranded DNA copy of the RNA genome and two long terminal repeats (LTRs)
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