Rapid identification of viable retrovirus-transduced cells using the green fluorescent protein as a marker

Various methods for determining the expression of the beta-galactosidase (beta-gal) gene after retroviral transduction were compared as a means to assess retroviral titre. To allow better comparison, different retroviral vectors were constructed carrying two mutants of the green fluorescent protein and assessed as sensitive markers of retroviral gene transfer. It could be demonstrated that GFP is generally superior to beta-gal in terms of sensitivity, speed and non-invasiveness of assay, allowing easy direct FACS sorting of populations of transduced cells. This opens the possibility of enrichment by sorting of ex vivo transduced cells in gene therapy protocols

Modulation of Moloney leukemia virus long terminal repeat transcriptional activity by the murine CD4 silencer in retroviral vectors

We investigated whether CD4 gene regulatory sequences might be useful for developing transcriptionally targeted Moloney murine leukemia virus (Mo-MLV)-based retroviral vectors for gene expression specifically in CD4(+) cells. We could modulate Mo-MLV long terminal repeat (LTR) activity by inserting a 438-bp-long fragment containing the murine CD4 silencer in the LTR of the vector; both beta-galactosidase and green fluorescent protein reporter gene activities were strongly down-regulated in both murine and human CD8(+) cells, but not in CD4(+) lymphoid cell lines and freshly isolated lymphocytes transduced with this vector, compared with the findings using a control vector carrying wild-type LTRs. Titration experiments on NIH-3T3 cells revealed that inclusion of the CD4 silencer in the LTRs did not reduce the titer of the vectors. These findings indicate that a cellular silencer can be successfully included in retroviral vectors, where it maintains its transcription-regulatory function, thus suggesting a novel approach to transcriptional targeting

Professor Barrie Vernon-Roberts, AO, MD, BSc, PhD, FRCPath, FRCPA, FAOrthA (Hon), FRS.SA

This issue of Inflammopharmacology contains papers that have been submitted to commemorate the life and work of Professor Barrie Vernon-Roberts, an outstanding clinical scientist in the field of bone pathology and its pharmacological regulation. This review briefly summarizes his major works and achievements as well as a list of his publications.K. D. Rainsford, D. R. Hayne