Helios(®) Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates.
The transfection of vertebrate inner ear hair cells has proven to be challenging. Therefore, many laboratories attempt to use and improve different transfection methods. Each method has its own advantages and disadvantages. A particular researcher's skills in addition to available equipment and the type of experiment (in vivo or in vitro) likely determine the transfection method of choice. Biolistic delivery of exogenous DNA, mRNA, or siRNA, also known as Helios(®) Gene Gun-mediated transfection, uses the mechanical energy of compressed helium gas to bombard tissue with micron- or submicron-sized DNA or RNA-coated gold particles, which can penetrate and transfect cells in vitro or in vivo. Helios(®) Gene Gun-mediated transfection has several advantages: (1) it is simple enough to learn in a short time; (2) it is designed to overcome cell barriers even as tough as plant cell membrane or stratum corneum in the epidermis; (3) it can transfect cells deep inside a tissue such as specific neurons within a brain slice; (4) it can accommodate mRNA, siRNA, or DNA practically of any size to be delivered; and (5) it works well with various cell types including non-dividing, terminally differentiated cells that are difficult to transfect, such as neurons or mammalian inner ear sensory hair cells. The latter advantage is particularly important for inner ear research. The disadvantages of this method are: (1) low efficiency of transfection due to many variables that have to be adjusted and (2) potential mechanical damage of the tissue if the biolistic shot parameters are not optimal. This chapter provides a step-by-step protocol and critical evaluation of the Bio-Rad Helios(®) Gene Gun transfection method used to deliver green fluorescent protein (GFP)-tagged full-length cDNAs of myosin 15a, whirlin, β-actin, and Clic5 into rodent hair cells of the postnatal inner ear sensory epithelia in culture.
Laboratory of Molecular Genetics, Section on Human Genetics, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, MD, 20892-3729, USA. belyants@nidcd.nih.gov.
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Belyantseva
D000199:Actins / Q000235:genetics; D000818:Animals; D019470:Biolistics / Q000295:instrumentation*; D018118:Chloride Channels / Q000235:genetics; D007758:Ear, Inner / Q000166:cytology* / Q000378:metabolism*; D004848:Epithelium; D018014:Gene Transfer Techniques / Q000295:instrumentation; D006046:Gold; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D009218:Myosins / Q000235:genetics; D009924:Organ Culture Techniques; D010316:Particle Size; D051381:Rats
D000199:Actins; D018118:Chloride Channels; D049452:Green Fluorescent Proteins; D006046:Gold; D009218:Myosins
Actin; Biolistic transfection; Clic5; GFP; Gene gun; Hair cell; Immunofluorescence; Inner ear; Myosin; Stereocilia; Whirlin