Ekaterina Karelina için film müziği, video kayıt, CAG-EGFP plazmid hazırlanması için 3D animasyon ve Dr. Peter Blaesse Ivan Molotkov yardım için teşekkür ederim. Finlandiya Uluslararası Hareketlilik, Finlandiya Kültür Vakfı ve Finlandiya Akademisi Merkezi hibe destek verdi.

1. Giriş Transgenik hayvanlar oluşturulması, genlerin fonksiyonlarının incelenmesi, canlı hayvanlar 1 ve unraveling hastalığın mekanizması 2,3 yanı sıra hücrelerin 4 özellikleri işlemek için güçlü bir yöntem. Ancak, prosedür, bu nedenle bu tür viral enjeksiyon 5, elektroporasyonu 6 ve utero elektroporasyon 7,8 neonatal ventriküler enjeksiyonu gibi alternatif gen teslimat yöntemleri kullanmak dair teminat, son derece zaman alıcı ve pahalı oldukça zahmetli. ;, Örneğin kortikal ilgi alanında yerel bir ifade deseni oluşturmak olanağı göçmen olmayan hücre tiplerinin yerinde transfeksiyon non-viral genetik yapıları kullanımı: doğum sonrası ventriküler olmayan enjeksiyon ve elektroporasyon yöntemi özel bir takım avantajları vardır astrositler. Bu video, CMV artırıcı (pCAG-EGFP) 9 tavuk beta aktin organizatörü altında gelişmiş yeşil flüoresan protein plazmid bir kodlama kullanılarak yenidoğan sıçan beyin, doğum sonrası gen teslim ayrıntılı bir prosedür ortaya koymaktadır. Biz ince cam pipetler stereotaktik bir cihaz üzerinde imalat ve montajı mikroenjektör plazmid içeren enjeksiyon solüsyonu hazırlanması göstermektedir. Sonra biz, enjeksiyon prosedür hakkında ve forseps elektrotlar ve in vivo porator kullanarak elektroporasyon hakkında, ameliyat gerçekleştirme hakkında izofluran ile sıçan yavrular bayıltmadan hakkında konuşmak . Son olarak, beklenen sonuçları, bakış açıları ve bu deneyler sırasında ortaya çıkabilecek zorlukları kısaca tartışacağız. 2. Elektroporasyon için Plazmid Hazırlık <ol><li> Biz genellikle 10 ul 200 ul ince duvar tüp plazmid enjeksiyon çözüm hazırlar. Bu çözüm çeşitli deneyler için yeterli olacaktır. </li><li> Biz 1 ul 10X PBS (Malzeme ve ekipman) ve% 0,1 &#39;Hızlı Yeşil su solüsyonu (Malzeme ve ekipman) 1 ul ul plazmid çözüm 8 (Malzeme ve ekipman) karıştırın. </li><li> Plazmid enjeksiyon çözüm nihai konsantrasyonu mg / ml 1 ile 3 arasında olmalıdır. DNA konsantrasyonu yüksek cam kapiller ince ucu ile enjeksiyon için çok viskoz ise plazmid DNA düşük konsantrasyonlarda, transfeksiyon verimliliği azaltacaktır. </li></ol> 3. Cam İğne Hazırlık <ol><li> Cam pipet hazırlanması için, biz, maksimum parametreleri çekerek ısıtma ve filament ile kılcal borosilikat cam (Malzeme ve ekipman) ve dik bir elektrot cam çektirmesi (Malzeme ve ekipman) kullanın. </li><li> Daha sonra ucu çapı 10-20 mikron ulaşmak için bir kağıt parçası kullanarak pipet ucu bölünürler. </li><li> Biz mikroskop amacı altında ucu çapı ve şekli kontrol edin. </li></ol> 4. Mikroenjektör Montaj <ol><li> Kılcal ince bir polimer (Malzeme ve ekipman) (Malzeme ve ekipman), mineral yağı ile (Malzeme ve ekipman) yaklaşık 1 / 3 dolgu hacminin 10 ul Hamilton şırınga kullanma. Hava kabarcıkları kaçının! </li><li> Sonra biz, mineral yağ ile cam pipet doldurmak ve Hamilton şırıngaya pipet yerleştirin. Hava kabarcıkları kaçının! </li><li> Cam pipet ile şırınga sonra (bkz. Malzeme ve ekipman) bir denetleyici bağlı mikroenjektör sabittir. </li><li> Stereotaktik aleti (Malzeme ve ekipman) sabit mikroenjeksiyon kurulum bize plazmid enjeksiyon solüsyonu ile güvenle cam pipet ucu tüpe imkanı bulursunuz. </li><li> Ucu çözüm yüzeye değdiğinde, 1 ile daha fazla ya da 2 mm daldırma ve mikro enjektör denetleyicisi kullanarak enjeksiyon karışımı yaklaşık 1 ul pipet doldurun. </li></ol> 5. Bayıltmadan hayvan Tüm işlemler burada sunulan hayvan deneyleri için Helsinki düzenlemelerin Üniversitesi göre yapıldı. <ol><li> Her deneme için izofluran yaklaşık 2 ml (Malzeme ve ekipman) ile gaz sızdırmaz bir şırınga (Malzeme ve ekipman) doldurun. </li><li> Şırınga, hava akımının kaynağına bağlı anestezi birimi (Malzeme ve ekipman) sabit değil, hayvan kutusu ve maske stereotaktik kurulum sabit. </li><li> Anestezi ünitesi, hava akımı yaklaşık 250 ml / dk ve izofluran düzeyi% 4&#39;e ayarlayın. </li><li> Biz, 2-5 dakika süreyle hayvan kutuya yavru sıçan yerleştirin. </li><li> Yavru durur taşırken, stereotaktik kurulum bağlı ısıtma yastığı (Malzeme ve ekipman) üzerine yerleştirin. </li><li> Kullanıcı yavru kafa rostral parçası bayıltmadan maske içine yerleştirin. </li><li> Için 5 ila 10 dakika sürerderin anestezi girmek için yavru. </li><li> Anestezi derinliği bir kuyruk tutam kontrol edin ve yaklaşık% 2,0-1,5 izofluran girişi azaltmak. </li></ol> 6. Cerrahi, Mikroenjeksiyon ve Elektroporasyon <ol><li> % 70 etanol ile yavru kafasına cilt davranın. </li><li> (Malzeme ve ekipman), küçük makas (Malzeme ve ekipman) ve ince forseps kullanarak, alnında yavru bir ense cilt kesti. </li><li> Yanlara doğru bükün ve deri parçaları, hafif baş ve cilt fiksasyon için kafatasının işitsel orifisleri kulak bar çekin. </li><li> Binoküler bir mikroskop kullanarak, kafatası bregma noktayı bulmak. </li><li> Stereotaktik koordinatları kullanarak ilgi bölge tanımlayın. </li><li> Bu bölgenin üstüne enjeksiyon pipet yerleştirin ve kafatası yüzeye enjeksiyon solüsyonu 25-50 nl bırakarak işaretlemek. </li><li> Delinmiş alanda sıvı görünene kadar nazik ve dikkatli bir kafatası, mikroskop altında, yüksek hızlı bir cerrahi matkap (Malzeme ve ekipman) ile işaretlenmiş bir noktada delin. </li><li> Daha iyi iletkenlik elde etmek için geçerli iletken jel ile kafatası tarafta bir electropotation forseps elektrotlar (Malzeme ve ekipman) (Malzeme ve ekipman) sabitleyin. </li><li> Küçük tampon kullanarak deliğe damla sıvı (Malzeme ve ekipman) çıkarın ve enjeksiyon yeri koordinatlarına göre delik cam iğne batırın. </li><li> 25 ile 100 nl 5 ila 20 nl / sn hızda enjeksiyon solüsyonu infüze. </li><li> Pipet hızla kaldırmak ve hemen electroporate. Elektroporasyon 1 Hz frekansta 99 V 50 ms ve genlik süresi beş dikdörtgen darbeler uygulayarak yapılır. </li><li> Elektrotlar ve kulak çubukları çıkartın. </li><li> Dilsiz (Malzeme ve ekipman) ve keskin bir forseps ve cerrahi konuları (Malzeme ve ekipman) bir kombinasyonu kullanılarak kesilmiş deri diker. </li><li> Anestezi sonrası kurtarma yardım etmek için 15-30 dakika sıcak odasına yavru yerleştirin. </li><li> Sonra yavru anne kafesine geri koymak. </li></ol> 7. Temsilcisi Sonuçlar Başarılı bir şekilde transfekte hücreler transgen ifadesi elektroporasyon yaklaşık 10 saat sonra görünür ve birkaç hafta boyunca sabit kalacaktır. Biz yukarıda anlatıldığı gibi derin kortikal katmanları veya postnatal günde 2 (P2) Wistar sıçan yavrular striatum bölge içine ya da plazmid microinjected ve in vivo elektroporasyon yapılan. Beyin dilimleri (kalınlığı 400 mikron) 2 ila 6 gün sonra kesilir (P4-P8) (HEPES Earle Dengeli Tuz Çözeltisi tamponlu) dilimleme soğuk ortamda 10 vibrotome kullanarak. Görüntü elde etme, oda sıcaklığında dilimleme orta Zeiss Axioplan 2 LSM5 Pascal konfokal mikroskop (Zeiss, Almanya) kullanılarak gerçekleştirildi. Derin kortikal katmanları ve striatumda enjeksiyon siteleri EGFP ifade çapı (Şekil 1A, B) yaklaşık 200-300 mikron kompakt bir bölgede bulunan birçok transfekte hücreleri içeriyordu. Transfekte hücreler içinde, EGFP ifade düzeyi, farklı şekillerde (Şekil 1D) ve akson demetleri (Şekil 1E) dikenler ile dendritler de dahil olmak üzere ince hücresel süreçler (Şekil 1C) görüntüleme izin için yeterince yüksek. EGFP elektroporasyon sonra hücre canlılığı yanı sıra sağlam bir istikrar aksonların distal parçaları gibi kortikal terminalleri (Şekil 1F) (1.5-2 mm hücre gövdeleri) izleme izin verdi. <img src="/files/ftp_upload/2244/2244fig1.jpg" alt="Şekil 1" /> Şekil 1 (A) başarıyla striatum bölgede plazmid enjeksiyon yeri tasvir P4 sıçan beyin hücreleri transfekte. (B) P5 sıçan beyin derin kortikal tabakalar halinde transfekte hücreleri (kortikal yüzey altında yaklaşık 1000 mm). (C) P8 sıçan beyin striatum bölgede transfekte hücresi. (D) P8 sıçan beyin striatum bölgede transfekte hücre Dendritik süreçler; oklar dendritik dikenler ve filopodia farklı türlerini belirtir. (E) teminat yolağı içinde transfekte hücrelerinin Aksonlar. (F) P8 sıçan korteks (ok ucu) pial yüzey sonlandırma Aksonlar. Ölçek çubukları, A, B, E ve F 100 mikron, 10 mikron, C ve D

<ol>
	<li>Gerlai, R., Clayton, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+hippocampal+function+in+transgenic+mice:+an+ethological+perspective.+Trends+Neurosci.">Analyzing hippocampal function in transgenic mice: an ethological perspective. Trends Neurosci.</a> 22, 47-51 (1999).</li><li>McGowan, E., Eriksen, J., Hutton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+modeling+Alzheimer's+disease+in+transgenic+mice.">A decade of modeling Alzheimer's disease in transgenic mice.</a> Trends Genet. 2, 281-289 (2006).</li><li>Cryan, J. F., Holmes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ascent+of+mouse:+advances+in+modeling+human+depression+and+anxiety.">The ascent of mouse: advances in modeling human depression and anxiety.</a> Nat. Rev. Drug Discov. 4, 775-790 (2005).</li><li>Wells, T., Carter, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+neural+function+in+transgenic+rodents:+towards+a+comprehensive+strategy.">Genetic engineering of neural function in transgenic rodents: towards a comprehensive strategy.</a> J. Neurosci. Methods. 108, 111-130 (2001).</li><li>Pilpel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=reproducible+transduction+of+select+forebrain+regions+by+targeted+recombinant+virus+injection+into+the+neonatal+mouse+brain.">reproducible transduction of select forebrain regions by targeted recombinant virus injection into the neonatal mouse brain.</a> J. Neurosci. Methods. 182, 55-63 (2009).</li><li>Boutin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+vivo+electroporation+of+the+postnatal+rodent+forebrain.">Efficient in vivo electroporation of the postnatal rodent forebrain.</a> PLoS One. 3, e1883-e1883 (2008).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev. Biol. 240, 237-246 (2001).</li><li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat. Protoc. 1, 1552-1558 (2006).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proc. Natl. Acad. Sci. U S A. 101, 16-22 (2004).</li><li>De Simoni, A., Yu, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+organotypic+hippocampal+slice+cultures:+interface+method.">Preparation of organotypic hippocampal slice cultures: interface method.</a> Nat. Protoc. 1, 1439-1445 (2006).</li><li>Walantus, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> J. Vis. Exp. , (2007).</li><li>Walantus, W., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E16+rat+embryos.">In utero intraventricular injection and electroporation of E16 rat embryos.</a> J. Vis. Exp. , (2007).</li><li>Umeshima, H., Hirano, T., Kengaku, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-based+nuclear+movement+occurs+independently+of+centrosome+positioning+in+migrating+neurons.">Microtubule-based nuclear movement occurs independently of centrosome positioning in migrating neurons.</a> Proc. Natl. Acad. Sci. U S A. 104, 16182-16187 (2007).</li><li>Ashwell, K., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Atlas of the Developing Rat Nervous System. , (2008).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (2007).</li></ol>

Yaşayan kemirgen beyin içine gen teslimat yöntemleri ve doğum sonrası elektroporasyon 6 için, son zamanlarda, utero elektroporasyon 7,8,11,12 kurulan ve. Ancak, bu yöntemlerin çeşitli uygulamalar için sınırlayıcı olabilir plazmid DNA, intraventriküler enjeksiyon dayanmaktadır. Örneğin, bu yöntemleri, hipokampus, ne de kortikal astrositler gibi göçmen olmayan hücre tiplerinin transfeksiyon gibi belirli beyin bölgelerindeki hücrelerini hedef izin vermez. Non-ventrikü...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2A-sa dumb Tweezers, 115mm</td>
<td>equipment</td>
<td>XYtronic</td>
<td>XY-2A-SA</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Biological Temperature Controller with stainless steel heating pad</td>
<td>equipment</td>
<td>Supertech</td>
<td>TMP-5b</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate tube with filament</td>
<td>material</td>
<td>Sutter Instruments</td>
<td>BF120-69-10</td>
<td>Glass needle</td>
</tr>
<tr>
<td>Disposable drills</td>
<td>material</td>
<td>Meisinger</td>
<td>HP 310 104 001 001 008</td>
<td>&nbsp;</td>
<tr>
<td>Dulbeco's PBS 10X</td>
<td>reagent</td>
<td>Sigma</td>
<td>D1408</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps, 110 mm</td>
<td>equipment</td>
<td>FST</td>
<td>91150-20</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Ealing microelectrode puller</td>
<td>equipment</td>
<td>Ealing</td>
<td>50-2013</td>
<td>Vertical electrode glass puller</td>
</tr>
<tr>
<td>Ethilon monofil polyamide 6-0 FS-3 16 mm 3/8c</td>
<td>material</td>
<td>Johnson &amp; Johnson Medical</td>
<td>EH7177H</td>
<td>Surgical threads</td>
</tr>
<tr>
<td>Exmire micro syringe 10.0 ml</td>
<td>equipment</td>
<td>Exmire</td>
<td>MS*GLLX00</td>
<td>Gas-tight syringe</td>
</tr>
<tr>
<td>Fast Green</td>
<td>reagent</td>
<td>Sigma</td>
<td>F7252</td>
<td>&nbsp;</td>
<tr>
<td>Forceps electrodes</td>
<td>equipment</td>
<td>BEX</td>
<td>LF650P3</td>
<td>Treat with 70% ethanol for disinfection prior to use</td>
</tr>
<tr>
<td>Foredom drill control</td>
<td>equipment</td>
<td>Foredom </td>
<td>FM3545</td>
<td>Surgical drill power supply and control. Currently available analogue is micromotor kit K.1070 (Foredom) </td>
</tr>
<tr>
<td>Foredom micro motor handpiece</td>
<td>equipment</td>
<td>Foredom</td>
<td>MH-145</td>
<td>Currently available analogue is micromotor kit K.1070 (Foredom)</td>
</tr>
<tr>
<td>Gas anesthesia platform for mice</td>
<td>equipment</td>
<td>Stoelting</td>
<td>50264</td>
<td>Assembled on stereotaxic instrument</td>
</tr>
<tr>
<td>Isoflurane</td>
<td>reagent</td>
<td>Baxter</td>
<td>FDG9623</td>
<td>&nbsp;</td>
<tr>
<td>Micro dressing forceps, 105 mm</td>
<td>equipment</td>
<td>Aesculap</td>
<td>BD302R</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Microfil</td>
<td>material</td>
<td>WPI</td>
<td>MF34G-5</td>
<td>Micro syringe filling capillaries</td>
</tr>
<tr>
<td>Mineral oil</td>
<td>reagent</td>
<td>Sigma</td>
<td>M8410</td>
<td>&nbsp;</td>
<tr>
<td>NanoFil Syringe 10 microliter</td>
<td>equipment</td>
<td>WPI</td>
<td>NANOFIL</td>
<td>Hamilton syringe</td>
</tr>
<tr>
<td>plasmid CAG-EGFP</td>
<td>reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Extracted and purified with EndoFree Plasmid Maxi Kit (Qiagen) and dissolved in nuclease free water to concentration 1.5 mg/ml</td>
</tr>
<tr>
<td>Pulse generator CUY21Vivo-SQ</td>
<td>equipment</td>
<td>BEX</td>
<td>CUY21Vivo-SQ</td>
<td>&nbsp;</td>
<tr>
<td>Schiller electrode gel</td>
<td>reagent</td>
<td>Schiller AG</td>
<td>2.158000</td>
<td>Conductive gel</td>
</tr>
<tr>
<td>Small animal stereotaxic instrument</td>
<td>equipment</td>
<td>David Kopf Instruments</td>
<td>900</td>
<td>&nbsp;</td>
<tr>
<td>Stoelting mouse and neonatal rat adaptor</td>
<td>equipment</td>
<td>Stoelting</td>
<td>51625</td>
<td>Assembled on stereotaxic instrument.
Treat earbars with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Student iris scissors, straight 11.5 cm</td>
<td>equipment</td>
<td>FST</td>
<td>91460-11</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Sugi absorbent swabs 17 x 8 mm</td>
<td>material</td>
<td>Kettenbach</td>
<td>31602</td>
<td>Surgical tampons</td>
</tr>
<tr>
<td>UMP3 microsyringe pump and Micro 4 microsyringe pump controller</td>
<td>equipment</td>
<td>WPI</td>
<td>UMP3-1</td>
<td>Microinjector and controller</td>
</tr>
<tr>
<td>Univentor 400 Anesthesia Unit</td>
<td>equipment</td>
<td>Univentor</td>
<td>8323001</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

gene delivery to postnatal rat brain by non-ventricular plasmid injection and electroporation

Transgenik hayvanlar oluşturulması, in vivo olarak ilgi çekici bir gen fonksiyonları okuyor standart bir yaklaşımdır. Ancak, birçok nakavt veya transgenik hayvanlar değiştirilmiş gen ifade ya da tüm organizmanın silinir bu durumda uygulanabilir değildir. Ayrıca, telafi edici mekanizmaların çeşitli genellikle sonuçları yorumlamak zor. Telafi edici etkileri ya zamanlama gen ekspresyonu veya transfekte hücrelerinin miktarını sınırlayarak kontrol altına alınabilir. Postnatal olmayan ventriküler mikroenjeksiyon ve in vivo elektroporasyon yöntemi, yeni doğan kemirgen beyin ilgi küçük bir bölgeye doğrudan genler, siRNA veya boya molekülleri hedeflenen teslim sağlar . Geleneksel ventrikül enjeksiyon tekniği aksine, bu yöntem, göçmen olmayan hücre tiplerinin transfeksiyon sağlar. Burada açıklanan yöntem vasıtasıyla transfekte Animals in vivo görüntüleme veya akut beyin dilimleri elektrofizyolojik deneylerde, örneğin, iki foton kullanılabilir.

Watch this Scientific Journal Video about Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation at JoVE.com

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Bu protokol, belli bir alanda yaşayan kemirgen beyin için genetik yapıları teslim olmayan bir viral yöntem açıklanır. Yöntemi plazmid hazırlanması, mikropipet imalat, yenidoğan sıçan yavru cerrahi, yapı mikroenjeksiyon, ve oluşur<em&gt; In vivo</em&gt; Elektroporasyon.

Non-ventriküler Plazmid Enjeksiyon ve Elektroporasyon Postnatal Rat Beyin Gen Teslimat

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

gene-delivery-to-postnatal-rat-brain-non-ventricular-plasmid

Research

JoVE Journal

Neuroscience

15.0K Görüntüleme.  University of Helsinki. Bu protokol, belli bir alanda yaşayan kemirgen beyin için genetik yapıları teslim olmayan bir viral yöntem açıklanır. Yöntemi plazmid hazırlanması, mikropipet imalat, yenidoğan sıçan yavru cerrahi, yapı mikroenjeksiyon, ve oluşur In vivo Elektroporasyon.

Video: Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In utero Elektroporasyon Kortikal nöronlar Altküme Gen Fonksiyonu Öğrenmenin İlköğretim Nöronal Kültür takip

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Nörobilim

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

Virüs tabanlı Cre Rekombinasyon kullanarak Postnatal Fare Beyin Gelişimi Gen Fonksiyonu Mosaic Analizi

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

Verimli kullanarak birden çok CNS Topraklar&#39;da Gen Teslimat In Utero Elektroporasyon

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Fare Utero Elektroporasyon: Kontrollü Spatiotemporal Gen Transfeksiyon

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Tarafından geliştirilmesi Fare İç Kulak için Gen Transferi<em&gt; In Vivo</em&gt; Elektroporasyon

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Tarafından Embriyonik ve Erişkin Nöral Kök Hücre Genişletme<em&gt; In Utero</em&gt; Elektroporasyon veya Viral Stereotaksik Enjeksiyon

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

Fare Akut Beyin Dilimleri Neuroblast Göç vivo Doğum Sonrası Elektroporasyonla ve Time-lapse Görüntüleme

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Kortikal internöronlar alt tip-selektif Elektroporasyon

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Ön Talamusun Çekirdek Derin Beyin Stimülasyon sonra Sıçan Hippocampus Gen İfade Değişim Analizi

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

Sıçan beyin dokusunda incelemek için THP kullanılarak hücre-tipi-spesifik gen ifadesi

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...