Tricky Transfections

A combination of microinjection and electroporation inserts genes into hard-to-reach cells.

By | March 1, 2015

© GEORGE RETSECK

The study of human hearing—how it develops, functions, and can diminish with age or disease—would be a great deal easier if only the hair cells of the ear weren’t so difficult to get at. These mechanosenory cells, which convert sound-wave vibrations into electrical signals for the auditory nerves, are buried deep within the organ of Corti in the cochlea and are largely inaccessible when it comes to transfecting genes for functional studies, even when the tissue is cultured.

Indeed, with the exception of laborious viral vector–based delivery, standard transfection approaches “just don’t work,” says hearing researcher Tony Ricci of Stanford University. But now, Ulrich Müller and his team from the Scripps Research Institute have found that by dissecting out the cochlear duct, injecting the genetic payload right next to the hair cells, and then applying an electric current to temporarily create pores in the hair cells’ plasma membranes (electroporation), they can succeed where other transfection methods have failed.

“It’s very exciting because it fills a gap, technically, in what we are able to do when trying to investigate and manipulate these auditory sensory cells,” says Ricci. For gene-function studies, he says, you often “had to make a mouse,” which can take up to a year. But now, “you can basically use ‘injectoporation’ as a tool for screening molecular interactions and then you can decide if you want to make a mouse later,” he says.

Müller has already used the technique to decipher components of the elusive hair-cell mechanotransduction machinery, but says that, in principle, injectoporation could be used for any tissue of the body “where access of the plasmid to the cells of interest is limited.” (Nature Protocols, 9:2438-49, 2014)

TECHNIQUE HOW IT WORKS PRODUCTION TIME MAXIMUM INSERT SIZE USE IN VIVO
Viral transfection Gene of interest is inserted into a viral vector, which is used to infect hair cells. Several weeks Typically 5 Kb Yes
Injectoporation DNA, RNA, or even proteins, in principle, are injected into the vicinity of hair cells in cultured cochlear-duct cells using a micropipette. Sample is then electroporated. Practically none. DNA can be loaded directly into pipette. Longest so far: 10 Kb No

 

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Comments

Avatar of: Brecht

Brecht

Posts: 1

March 12, 2015

Wow, why not developing this approach for in vivo situation!

Avatar of: PR Prof

PR Prof

Posts: 2

March 12, 2015

This technique has actually been around for some time now.  (I'm surprised this is the first time electroporation has been tried for these cells, but kudos to the authors for getting it to work.)   I tried using this technique several years ago during my Ph.D. project on human neutrophils, but it still proved to be quite difficult to achieve reliable results on those cells.

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