Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries
Final rept. 1 Jul 2010-31 Dec 2012
CLEVELAND CLINIC FOUNDATION 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 252 puromycin resistant 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 78 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 78 clones. DNA sequencing has identified the cDNA inserts. These inserts were expressed in na ve MDS cell pools. Eight of these cDNAs were validated they induced myeloid colonies in vitro and engrafted in the marrow of SG3, but not NSG mice.
- Medicine and Medical Research