|M.Sc Student||Strulovici Yael|
|Subject||Genetic Manipulations of Human Embryonic Stem Cells|
|Department||Department of Medicine||Supervisors||PROFESSOR EMERITUS Joseph Itskovitz|
|DR. Hani Segev|
|Full Thesis text|
Human embryonic stem cells (hESCs) are pluripotent cells derived from embryos created for IVF purposes and donated to science. Thanks to the hESCs ability of self-renewal and to their potential to differentiate into any cell type, they are widely used as a model for studying genes function and developmental pathways during normal and abnormal embryogenesis. The cells can also be used for the study of pathogenesis, for drug testing and as an unlimited source of tissues for transplantation, as therapy for diseases caused by the dysfunction or degradation of a limited number of specific cell types, such as Parkinson’s disease and diabetes.
However, these applications rely on the ability to establish a homogenous population of differentiated cells and, to date, no differentiation protocol has lead to a uniform population of cells, but instead, to a mixed population of cells at different differentiation levels of different cell types. One way to purify the heterogeneous population of cells is to genetically manipulate the cells to carry selectable markers of the desired cell type. This can be achieved transiently or stably by introducing a vector containing selectable markers into the targeted cells. Homologous recombination (HR) can be used to introduce a vector into a specific site on the targeted cells genome in order to knock in or knock out a specific gene.
We successfully homologous recombined hESCs with a plasmid containing selectable markers under a stemness gene promoter using TransIT-LT1, a polyamine transfection reagent never before published to transfect hESCs. We have obtained clones at higher success rate than previously published for homologous recombination of hESCs and further proliferated them into individual cell lines.
We also established a protocol for HR of hESCs using bacterial artificial chromosomes (BACs), that are large vectors, never before used to transfect hESCs, and used it to obtain clones stably marked for a stemness gene. We then used this protocol to successfully target hESCs with a neural marker-derived BAC. The obtained clones were further propagated and differentiated into neurons that can be easily identified using the selectable markers integrated in their genome for future use in cell migration, transplantation and mutation studies.