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line to generate polyclonal as well as single clone retroviral producer populations.
Vesicular Stomatitis Virus-G Protein (VSV-G) pseudotyped retroparticles were
produced and used to transduce the Jurkat T-cell line and the ZAP-70-deficient
Jurkat P116 T-cell line. Since recombinant retroviral vectors can be be susceptible
to rearrangements prior to their final integration as a DNA proviral genome, we
characterized the integrated conformation in transduced target cells by Southern
Blot analysis (Figure 2C). Genomic DNA was extracted from null & from trans-
duced Jurkat cells and P116 cells, digested with KpnI and probed with [P32]-
labeled DNA sequences complementary to the GFP reporter cDNA. We detected
DNA bands consistent with the predicted sized fragment expected from KpnI
digest of integrated unrearranged proviral DNA.
Figure 2. Design of the SINIL-2pr retrovector. A. pGFP/TKfus plasmid bears a full-length 3'LTR that
incorporates all the promoter and enhancer machinery intrinsic to the wild-type MoMLV retrovirus. The CMV
promoter element which substitutes the U3 region in the 5' LTR drives the expression of the retroviral genome
in transfected packaging cells for the production of replication-defective retrovirus. B. SINIL-2 pr retrovector
is designed by creating an NheI-AscI deletion to the 3'LTR of pGFP/TKfus and by replacing the U3 with
the full-length human IL-2 promoter which results in a self-inactivating retrovector whereby expression of the
EGFP and HSVTK fusion protein is dependent on the IL-2 promoter in cells transduced with SINIL-2pr
retroparticles. C. Southern Blot analysis of the integrated proviral DNA in Jurkat and p116 cells transduced
with SINIL-2pr retrovector at similar MOI. Genomic DNA was extracted from transduced and control null
cells, digested with KpnI and probed with [P32]-labeled probe complementary to the GFP reporter cDNA.
The detected DNA bands are consistent with the predicted sized fragment indicating no rearrangement in the
integrated proviral DNA.
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