Rapid and efficient genetic manipulation of gyrencephalic carnivores using in utero electroporation

Abstract

BackgroundHigher mammals such as primates and carnivores have highly developed unique brain structures such as the ocular dominance columns in the visual cortex, and the gyrus and outer subventricular zone of the cerebral cortex. However, our molecular understanding of the formation, function and diseases of these structures is still limited, mainly because genetic manipulations that can be applied to higher mammals are still poorly available.ResultsHere we developed and validated a rapid and efficient technique that enables genetic manipulations in the brain of gyrencephalic carnivores using in utero electroporation. Transgene-expressing ferret babies were obtained within a few weeks after electroporation. GFP expression was detectable in the embryo and was observed at least 2 months after birth. Our technique was useful for expressing transgenes in both superficial and deep cortical neurons, and for examining the dendritic morphologies and axonal trajectories of GFP-expressing neurons in ferrets. Furthermore, multiple genes were efficiently co-expressed in the same neurons.ConclusionOur method promises to be a powerful tool for investigating the fundamental mechanisms underlying the development, function and pathophysiology of brain structures which are unique to higher mammals.

In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation

“…We recently established and reported in utero electroporation procedure for ferrets and successfully expressed GFP in post-mitotic neurons of the cerebral cortex (Kawasaki et al, 2012). We therefore assumed that our procedure is also applicable to manipulation of gene expression in the OSVZ.…”

“…Ferrets were maintained as described previously (Kawasaki et al, 2004; Iwai and Kawasaki, 2009; Iwai et al, 2012; Kawasaki et al, 2012). The day of birth was counted as postnatal day 0 (P0).…”

“…By modifying the procedure for in utero electroporation of rodents, we recently created a procedure for in utero electroporation to express transgenes in post-mitotic neurons of the ferret cerebral cortex (Kawasaki et al, 2012). Briefly, pregnant ferrets were anesthetized with sodium pentobarbital, and their body temperature was monitored and maintained using a heating pad.…”

“…In utero electroporation is a useful technique to express genes of interest in the living rodent brain (Fukuchi-Shimogori and Grove, 2001; Saito and Nakatsuji, 2001; Tabata and Nakajima, 2001; Sehara et al, 2010; Ako et al, 2011), but successful application of in utero electroporation in higher mammals had not been achieved. Therefore, we recently developed and validated a rapid and efficient procedure for in utero electroporation in gyrencephalic carnivore ferrets and demonstrated that genes of interest can be efficiently expressed in post-mitotic neurons (Kawasaki et al, 2012). Here we show that transgenes can be also efficiently expressed in neural progenitors in germinal zones including the OSVZ using in utero electroporation in the developing ferret.…”

Microinjection of membrane-impermeable molecules into single neural stem cells in brain tissue

“…Electroporation allows for genetic manipulation of neural stem and progenitors cells in a relatively short time window and is therefore less time consuming than transgenesis [4][5][6] . In addition, electroporation of the developing neural tube is very versatile: it can be adapted to different species (mouse, rat and ferret [7][8][9] ) and allows for different types of manipulation, ranging from overexpression of a specific gene to gene knockdown 5 . As electroporation relies on the application of an electric field, only charged molecules can be delivered, thus limiting the spectrum of molecules that can be used.…”