טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAtar Orna
SubjectThe Use of RNAi Silence Transcription Factors Essential for
the Immune System and the Effect on Cancer
Development - PU.1 Proto-oncogene as a
Model
DepartmentDepartment of Biotechnology
Supervisor Professor Doron Melamed


Abstract

This thesis describes the construction of various siRNA constructs that are directed against PU.1. One of these constructs was very effective exhibiting long lasting silencing of PU.1 (pSuper-PU.1). We have developed a rapid and an easy assay for selecting the most efficient siRNA. The effectively of the pSuper-PU.1 was demonstrated by transient reporter gene assay and by silencing ectopic expression of PU.1. Expression levels of a reporter gene driven by Bcl-XL or Nramp1 promoters, genes that are known to be regulated by PU.1, were efficiently suppressed by pSuper-PU.1 in a macrophage cell line.
Attempts to stably express pSuper-PU.1 in a macrophage cell line lead to a dramatic growth inhibition, similar to the growth inhibition observed earlier in the lab by using a dominant negative IRF8 construct in hematopoietic cells. We found that this growth inhibition is probably due to G2/M arrest. In other words, knocking down PU.1 in macrophage cell line disrupted the delicate balance between factors promoting or blocking cell-cycle progression, resulting in overall growth inhibition and G2/M arrest.
PU.1 also has a pivotal role in the development of Friend erythroleukemia disease, which is characterized by proliferation and differentiation arrest of proerythrocytes. Friend erythroleukemia has provided a powerful tool for the study of multistage carcinogenesis e.g. leukemogenesis. We have used transformed murine erythroleukemia (MEL) cells, isolated from Friend virus-infected mice. Using pSuper-PU.1 for silencing PU.1, we were able to demonstrate an inhibition of the cell proliferation and a restoration in the ability of the proerythroblastic cells to spontaneously differentiate to mature erythroblasts. The data suggest that overexpression of PU.1 gene is involve in maintaining the leukemic phenotype of the disease by keeping the self renewal capacity of transformed erythroblastic cells and by blocking the terminal differentiation program towards erythrocytes.