Investigating Alternative Methods of Nucleic Acid Delivery in Oocytes

polycation-mediated delivery;oocytes;polyethyleneimine;RNAi;Ensa;Biochemistry

Ramachandran, AnitaMiller, Paul S. ;

Johns Hopkins University

关键词: polycation-mediated delivery;    oocytes;    polyethyleneimine;    RNAi;    Ensa;    Biochemistry;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/37254/RAMACHANDRAN-THESIS-2014.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Studies using RNA interference in oocytes have elucidated processes relating to oocyte function and fertilization. Currently, the only viable method for genetic manipulation of oocytes is microinjection, a very time consuming procedure. There is no quantitative evidence of successful transfections with lipid-based vehicles in oocytes with the zona pellucida intact. Polycations are effective vehicles for delivering nucleic acids into mammalian cells, but have not been tested in oocytes. I investigated whether polycation-mediated nucleic acid delivery is a feasible alternative to microinjection in oocytes. Polyethyleneimine (PEI) is a highly efficient polycationic vector that has been shown to deliver nucleic acids into a variety of cell types, including NIH 3T3 cells. PEI is commonly conjugated to polyethylene glycol (PEG) to reduce cytotoxicity and improve transfection efficiency. Commercial polycation-based nanoparticles, such as HappyFect, are also available for transfections in different cell types. As a functional assay to determine whether the polycations can be used as delivery agents in oocytes, I tested their ability to deliver siRNA that targets a specific gene called Ensa. I synthesized PEGylated PEI, characterized it using dynamic light scattering, and examined the ability of unmodified PEI and PEGylated PEI to complex with plasmid DNA or an RNA duplex using gel mobility shift assays. To assess the ability of PEI, PEGylated PEI and HappyFect to knockdown Ensa mRNA and ENSA protein in NIH 3T3 cells, I used an Ensa specific siRNA cocktail that was previously shown in the Evans Lab to knock down Ensa in oocytes via microinjection. Lipofectamine 2000 was used as a positive control, since it was shown to effectively knock down Ensa mRNA and ENSA protein in NIH 3T3 cells.Since the commercial polycation, HappyFect, was able to knock down Ensa transcript levels in NIH 3T3 cells, the delivery potential of HappyFect was then explored in oocytes.