الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 146 |  |  |
| | | from | 146 | | |
Abstract
chronic myeloid leukemia is a hematological malignancy characterized by recurrent chromosomal aberration which is the bcr-abl translocation found on the Philadelphia chromosome. This translocation originates mainly in the hematopoietic stem cells and plays an important role in the CML pathogenesis .
Novel treatment approaches were tried in a way to eradicate the CML stem cells. One of those approaches was the antibody-based therapy targeting the CML stem cells. A new target was discovered which is the IL1RAP , a toll like receptor , that was highly expressed on the cell membrane of the Philadelphia positive CML stem cells(Jaras et al.,2010) of CML patients and in hematopoietic CD34+ stem cells retrovirally transduced.
That finding was not supported by other researches and thus further studies were recommended. In this study , our primary goal was to evaluate the expression of IL1RAP gene in Philadelphia positive CD34+ hematopoietic stem cells which were prepared by the nucleofection of the bcr-abl gene into purified CD34+ hematopoietic stem cells .
The secondary goal was to evaluate the efficiency of the nucleofection , a modified electroporation technique , in the process of gene insertion .
In order to evaluate the expression of the IL1RAP gene in the UCB CD34+ cells transfected with the bcr-abl gene compared to those not transfected with the bcr-abl gene , five UCB samples were collected from five mothers .Each UCB sample yielded 10x106 purified small lymphocyte-like CD34+ hematopoietic stem cells .
The yield of each sample was divided into five parts. Each part consisted of 2 x106 purified CD34+ hematopoietic stem cells and was used as a sample for nucleofection. Thus, the five UCB samples provided 25 samples ready for nucleofection .
The plasmid carrying the desired gene fragment bcr-abl (P210 pcDNA3 ) was recovered from the bacterial stab according to the supplier protocols. Then it was verified for the presence of the gene of interest by digestion with the EcoRI restriction enzyme and visualized after gel electrophoresis of the cut bands.
After plasmid isolation, purification and verification , the umbilical cord blood hematopoietic CD34+stem cells were collected and prepared for the nucleofection process.
Transfection of the CD34+ stem cells was achieved by nucleofection which was followed by antibiotic selection of the transfected antibiotic resistant clones.
Post-nucleofection ,five test samples were harvested for real time PCR analysis of IL1RAP gene expression. The sixth sample was subjected to G418 antibiotic selection of the resistant clones. Then relative gene expression was assessed in the resistant clones using the reverse transcription quantitative real time PCR (RT-qPCR).
The success of the nucleofection process was evaluated by the successful nucleofection of pmax-GFP plasmid into the purified CD34+ hematopoietic stem cells ( green fluorescent signals from nucleofected CD34+ with pmax-GFP plasmid ) .
The IL1RAP gene expression levels of nucleofected cells ,48 hours post nucleofection , ranged from two to nine folds change.
The IL1RAP gene expression levels in subcultured G418 selected nucleofected cells , ranged from 4 to 11 folds change
Further studies should be conducted in order to identify the exact relationship between IL1RAP gene over expression and the expression of the bcr-abl gene.
Nucleofection , the modified electroporation technique , can be used as a safe and efficient gene insertion method especially in laboratories not equipped for viral transfection .