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(ii) Generation of induced pluripotent stem cells (iPSCs)

Induced pluripotent stem cells (iPSCs) are generated from a patient's own differentiated somatic cells which hold a good prospect in the field of regenerative medicine. Retroviral vectors have been used successfully which involves delivery of reprogramming factors. But around 20% of the chimeric offspring developed tumors due to re-activation of the c-myc oncogene. These chimeric offsprings were obtained from germline transmission of clones which were retrovirally reprogrammed. It has been found out that the ectopic expression of the reprogrammed factors have lead to formation of tumors and skin dysplasia. One of the ways to prevent the use of viral delivery system is by delivering the programming factors as recombinant proteins or by repeated plasmid transfections. But both of these methods have been proved as inefficient and slow. Since transposons have higher gene delivery efficiency and have the ability to get excised from the cells after reprogramming and differentiation hence they make a good choice for generating iPSCs. Transfection of somatic cells with piggyBac transposons carrying reprogramming factors and transposase from which reprogrammed iPSCs are selected and propagated to obtain individual iPSCs clones. Re-expression of transposase is done to remove the reprogramming factors in order to generate transgene-free iPSCs. This whole process is followed by negative selection to identify iPSCs which are transgene-free.

Since piggyBac system do not cause “foot print” mutation, it is therefore suitable for undergoing precise excision. Whereas, sleeping beauty system leaves behind altered insertion sites due to improper excision. Transgene-free iPSCs have been generated from both mouse and human embryonic fibroblasts from piggyBac system and its efficiency is comparable to retroviral vectors. Successful reprogramming of murine tail tip fibroblasts into fully differentiated melanocytes is being done using piggyBac system. piggyBac reprogramming system is now found to be more stable and quicker than lentiviral system.

(iii) Genetic modification of stem cells

Human embryonic stem cells are genetically modified using transposons. Even to insert bacterial artificial chromosomes (BACs) in human embryonic stem cells transposons are being used. To genetically modify hematopoietic stem cells both sleeping beauty and piggyBac are used. For permanent (or reversible in case of piggyBac ) genetic modifications of various types of stem cell transposons provide an effective mechanism for further use in gene therapy.