Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries
Annual rept. 1 Jul 2010-30 Jun 2011
CLEVELAND CLINIC OH
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We have successfully prepared sense expression libraries, characterized the size of their inserts which are fused to a green fluorescent protein tag, and packaged these libraries into lentivirus. Four different human MDS bone marrow isolates have been transfected with lentivirus at multiplicity of infection of 0.1. A collection of 250 puromycin clones were isolated, genomic DNA was prepared, and clones were screened for ability to undergo myeloid differentiation in response to GM-CSF. Only 30 of the puromycin resistant clones 80 acquired this phenotype. Transfection of MDS cells with antisense libraries did not generate any clones that acquired the desired differentiative phenotype. PCR was used to identify presence of the trans gene in the 80 clones. DNA sequencing is currently being performed to identify the inserts. Lentivirus is being prepared to express these inserts in na ve MDS cell pools. This validation will ensure that the insert causes the desired phenotype.
- Medicine and Medical Research