Search

Efficient, facile gene modification of cells has become an indispensable part of modern molecular biology. For the majority of cell lines and several primary populations, such modifications can be readily performed through a variety of methods. However, many primary cell lines such as stem cells frequently suffer from poor transfection efficiency. Though several physical approaches have been introduced to circumvent these issues, they often require expensive/specialized equipment and/or consumables, utilize substantial cell numbers and often still suffer from poor efficiency. Viral methods are capable of transducing difficult cellular populations, however such methods can be time consuming for large arrays of gene targets, present biohazard concerns, and result in expression of viral proteins; issues of concern for certain experimental approaches. We report here a widely applicable, low-cost (< $100 CAD) method of electroporation, applicable to small (1–10 μl) cell volumes and composed of equipment readily available to the average investigator. Using this system we observe a sixfold increase in transfection efficiency in embryonic stem cell lines compared to commercial devices. Due to efficiency gains and reductions in volume and applied voltage, this process improves the survival of sensitive stem cell populations while reducing reagent requirements for protocols such as Cas9/gRNAs transfections. [ABSTRACT FROM AUTHOR]

Of all methods exercised in modern molecular biology, modification of cellular properties through the introduction or removal of nucleic acids is one of the most fundamental. As such, several methods have arisen to promote this process; these include the condensation of nucleic acids with calcium, polyethylenimine or modified lipids, electroporation, viral production, biolistics, and microinjection. An ideal transfection method would be (1) low cost, (2) exhibit high levels of biological safety, (3) offer improved efficacy over existing methods, (4) lack requirements for ongoing consumables, (5) work efficiently at any scale, (6) work efficiently on cells that are difficult to transfect by other methods, and (7) be capable of utilizing the widest array of existing genetic resources to facilitate its utility in research, biotechnical and clinical settings. To address such issues, we describe here Pressure-jump-poration (PJP), a method using rapid depressurization to transfect even difficult to modify primary cell types such as embryonic stem cells. The results demonstrate that PJP can be used to introduce an array of genetic modifiers in a safe, sterile manner. Finally, PJP-induced transfection in primary versus transformed cells reveals a surprising dichotomy between these classes which may provide further insight into the process of cellular transformation. [ABSTRACT FROM AUTHOR]