본 연구는 서울 대학교 연구 그랜트 (800-20140542)에 의해 지원되었다, NRF / 교육 과학 기술부의 개척자 연구 프로그램 (2012-0009544)과 NRF / 교육 과학 기술부의 바이오 신호 분석 기술 혁신 프로그램 (2009-0090895) 과 NRF / 교육 과학 기술부의 그랜트 (2015M3A9E6028949).

동물에 대한 모든 실험은 안과 및 비전 연구에서 동물의 사용에 대한 비전 및 안과 문에 연구를위한 협회에 따라 수행하고, 지침과 규정은 서울 대학교 기관 동물 관리 및 사용위원회와 서울 대학교가 정한했다 대학 병원 바이오 안전성위원회. 1. 주입 키트 및 바이러스 성 벡터를 준비 <ol><li> 에틸렌 옥사이드 가스를 사용하여 멸균 33 G 무딘 바늘을 갖춘 마이크로 리터 주사기를 준비한다. 마이크로 튜브 (즉, 1 × 6 TU / μL)의 적절한 역가에 대한 PBS에서 바이러스 벡터를 희석. 주사기 임의 사강을 제거하는 주사기를 바이러스 벡터로 수회 세척. </li></ol> 바이러스 성 벡터의 2. 망막 주입 <ol><li> tiletami의 혼합물의 복강 내 주사 - (8 주 오래 즉, 6) 성인 쥐를 마취NE 및 zolazepam (1 : 1, 2.25 ㎎ / ㎏ 체중)과 자일 라진 하이드로 클로라이드 (0.7 ㎎ / ㎏ 체중) 또는 다른 적절한 마취 정권. </li><li> 페닐에 0.5 %의 점안제와 동공 팽창 및 0.5 % tropicamide. </li><li> 바이러스 벡터의 2 μL - 1.5로드하여 마이크로 리터 주사기를 준비합니다. </li><li> 눈꺼풀을 열고 편리 주입 적도 노출 및 운영 현미경에 초점을 눈 돌출. 주입 동안 발생할 수있는 바늘의 주입, 또는 변위 마무리까지 돌출 눈 위치를 유지한다. 단단히 안구를 유지하기 위해, 궤도 림 외부에서 손가락을 놓습니다. </li><li> 각막 표면 안과 점탄성 용액 한 방울을 적용한다. </li><li> 망막을 시각화하는 각막의 상단에 작은 원형 커버 슬라이드를 놓습니다. </li><li> 추가적인 망막 주 사용 멸균 1/2 30 G 바늘을 사용 윤부에 약간 후방에 작은 구멍이 천공. FO 구멍이 열등 확인R 우안과 편의상 좌안 우수한. 주 : 구멍 시간적 또는 비강에서 이루어진다면, 그것은 주입 후 눈꺼풀에 의해 커버되기 어렵다. 초기 구멍은 약간 만든 윤부를 따라 실행하고 쉽게 인식 할 수 윤부 혈관을 피하기 위해 윤부에 후부해야합니다. 초기 구멍을하는 동안 바늘로 렌즈를 타격 않도록주의하십시오. 렌즈 구멍을 피하기 위해 바늘의 전체 경사를 삽입하지 마십시오. </li><li> 사전 천공 구멍을 통해 마이크로 리터 주사기의 33 G 무딘 바늘을 놓고 가벼운 저항이 느껴지는 경우 지점까지 망막 하 공간에 바늘을 접근. 참고 : 망막 주사를 들어, 바늘의 가장 좋은 방법 각도는 조리개면에 대해 약 45도이며, 무딘 바늘은 유두 주위 지역을 향해 후방으로 밀어해야합니다. 점선 사각형은 망막 injectio의 유리 체강에서 제안 된 바늘의 경로를 표시그림 1에서 N. 참고 : 그들은 매우 부드럽고으로 관통 (또는 전달) 망막 층 경우에는 저항이 느끼지있을 것이다. 따라서, 온화한 저항 제 느낌 바늘 이미 RPE 층을 터치하고 바늘의 삽입이 중단되어야 함을 나타낸다. 바늘이 잠재적 인 망막 공간에 배치해야하기 때문에 과도한 압력과 공막 조직에 침투하지 않도록주의하십시오. 바늘이 과도한 전력에 의해 공막 조직을 천공하면, 저항없이 안구 궤도 공간으로 들어간다. (이 단계는 매우 중요하다) </li></ol><img alt="그림 1" src="/files/ftp_upload/53030/53030fig1.jpg" /> 망막 사출 그림 1. 회로도. H &amp; E 표시 망막 주사 바늘 경로와 구조를 묘사 마우스 눈의 스테인드 단면 (점선 사각형). 배율 : 40X, 스케일 바 :<html> 500 μm의. <a href="https://www.jove.com/files/ftp_upload/53030/53030fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <ol start="9"><li> 불필요한 조직 손상을 방지하고 조심스럽게 바늘을 철회 떨림없이 망막 하 공간에 부드럽게 바이러스 벡터 (즉, 1 × 6 TU / μL)를 주입한다. 2.4에 설명 된대로 주입하는 동안 단단히 안구를 잡습니다. </li><li> 더 망막 출혈이없는 것을 확인하기 위해 운영 현미경으로 주사 후 망막 여과포의 형성을 관찰한다. </li></ol><img alt="그림 2" src="/files/ftp_upload/53030/53030fig2.jpg" /> 그림 2. 망막 출혈없이 망막 Belb 형성. 운영 현미경으로 망막 주사 후 망막 belb의 현미경보기 망막 출혈없이 성공적으로 여과포 형성을 보여줍니다.M / 파일 / ftp_upload / 53030 / 53030fig2large.jpg &quot;대상 =&quot;_ 빈 &quot;&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오. <ol start="11"><li> 부드럽게 자체 밀봉 용 주사 부위를 덮도록 눈꺼풀을 닫을. 홈 케이지에 마우스를 반환하고, 평가까지 살아 유지. 참고 : 연구자가 절차에 익숙해 후, 다음 단계 건너 뛰어 신속한 절차에 반복 가능한 결과를 얻을 수 (2.5, 6, 10) 수 </li></ol> 망막 색소 상피의 유전자 전달의 효능 3. 평가 <ol start="1"><li> CO 2 챔버에서 쥐를 희생. 가위를 사용하여 눈을 명백히하다. </li><li> 며칠에 최소 1 시간 동안 4 ℃에서 4 % 파라 포름 알데히드 용액에서 쳐다. </li><li> 미세 집게로 각막을 잡고 마이크로 가위로 각막에 구멍. 윤부를 따라 가위질 한 후 각막을 제거합니다. 집게로 렌즈를 제거합니다. </li><li> M와 모양체 트림망막 박리를 허용 icro 가위. 조심스럽게 RPE / 맥락막 / 공막 단지에서 전체 망막을 잡아 당깁니다. 평면 실장 용 4~8 잎 있도록 시신경 수회 가까운 중심 둘레로부터 반경 RPE / 맥락막 / 공막 복합체를 잘라. </li><li> 아래 망막 색소 상피면을 만들고, 근육, 결막 및 시신경을 포함하는 공막 측에 남아있는 모든 조직을 손질 할 수있는 RPE / 맥락막 / 공막 복잡한 플립. 나머지 조직은 복잡한 요철 구조를 만들기 때문에이 단계는 플랫 RPE / 맥락막 / 공막 복합체를 확인하는 것이 중요하다. </li><li> (자세한 방법에 대한 내용을 참조, 선택 사항) 특정 연구 목적으로 기본 및 보조 항체 복합체를 품어. 유리 슬라이드에 RPE / 맥락막 / 공막 단지를 플랫 마운트 흡수성 스폰지로 PBS를 흡수한다. </li><li> 장착 솔루션의 30 μl를 추가하고 커버 슬라이드를 배치합니다. 형광 현미경 벡터 전달의 효능에 대한 샘플을 관찰한다. 에 샘플을 유지더 관찰 냉장고. </li></ol>

<ol>
	<li>Maguire, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficacy+of+gene+transfer+for+Leber's+congenital+amaurosis.">Safety and efficacy of gene transfer for Leber's congenital amaurosis.</a> N Engl J Med. 358 (21), 2240-2248 (2008).</li><li>Jacobson, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+leber+congenital+amaurosis+caused+by+RPE65+mutations:+safety+and+efficacy+in+15+children+and+adults+followed+up+to+3+years.">Gene therapy for leber congenital amaurosis caused by RPE65 mutations: safety and efficacy in 15 children and adults followed up to 3 years.</a> Arch Ophthalmol. 130 (1), 9-24 (2012).</li><li>Conlon, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+potency+and+safety+studies+of+an+AAV2-mediated+gene+therapy+vector+for+the+treatment+of+MERTK+associated+retinitis+pigmentosa.">Preclinical potency and safety studies of an AAV2-mediated gene therapy vector for the treatment of MERTK associated retinitis pigmentosa.</a> Hum Gene Ther Clin Dev. 24 (1), 23-28 (2013).</li><li>MacLaren, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+gene+therapy+in+patients+with+choroideremia:+initial+findings+from+a+phase+1/2+clinical+trial.">Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial.</a> Lancet. 383 (9923), 1129-1137 (2014).</li><li>Trapani, I., Puppo, A., Auricchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vector+platforms+for+gene+therapy+of+inherited+retinopathies.">Vector platforms for gene therapy of inherited retinopathies.</a> Prog Retin Eye Res. 43, 108-128 (2014).</li><li>Liang, F. Q., Anand, V., Maguire, A. M., Bennett, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraocular+delivery+of+recombinant+virus.">Intraocular delivery of recombinant virus.</a> Methods Mol Med. 47, 125-139 (2001).</li><li>Peyman, G. A., Lad, E. M., Moshfeghi, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravitreal+injection+of+therapeutic+agents.">Intravitreal injection of therapeutic agents.</a> Retina. 29 (7), 875-912 (2009).</li><li>Matsumoto, H., Miller, J. W., Vavvas, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+detachment+model+in+rodents+by+subretinal+injection+of+sodium+hyaluronate.">Retinal detachment model in rodents by subretinal injection of sodium hyaluronate.</a> J Vis Exp. (79), (2013).</li><li>Wert, K. J., Skeie, J. M., Davis, R. J., Tsang, S. H., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+injection+of+gene+therapy+vectors+and+stem+cells+in+the+perinatal+mouse+eye.">Subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye.</a> J Vis Exp. (69), (2012).</li><li>Eberle, D., Santos-Ferreira, T., Grahl, S., Ader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+transplantation+of+MACS+purified+photoreceptor+precursor+cells+into+the+adult+mouse+retina.">Subretinal transplantation of MACS purified photoreceptor precursor cells into the adult mouse retina.</a> J Vis Exp. (84), e50932 (2014).</li><li>Sahel, J. A., Roska, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+blindness.">Gene therapy for blindness.</a> Annu Rev Neurosci. 36, 467-488 (2013).</li><li>Allocca, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+adeno-associated+virus+serotypes+efficiently+transduce+murine+photoreceptors.">Novel adeno-associated virus serotypes efficiently transduce murine photoreceptors.</a> J Virol. 81 (20), 11372-11380 (2007).</li><li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+transduction+of+ocular+cells+with+a+bovine+immunodeficiency+viral+vector.">Sustained transduction of ocular cells with a bovine immunodeficiency viral vector.</a> Hum Gene Ther. 13 (11), 1305-1316 (2002).</li><li>Rolling, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapeutic+prospects+in+early+onset+of+severe+retinal+dystrophy:+restoration+of+vision+in+RPE65+Briard+dogs+using+an+AAV+serotype+4+vector+that+specifically+targets+the+retinal+pigmented+epithelium.">Gene therapeutic prospects in early onset of severe retinal dystrophy: restoration of vision in RPE65 Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium.</a> Bull Mem Acad R Med Belg. 161 (10-12), 497-508 (2006).</li><li>Le Meur, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+vision+in+RPE65-deficient+Briard+dogs+using+an+AAV+serotype+4+vector+that+specifically+targets+the+retinal+pigmented+epithelium.">Restoration of vision in RPE65-deficient Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium.</a> Gene Ther. 14 (4), 292-303 (2007).</li><li>Alexander, J. J., Hauswirth, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+viral+vectors+and+the+retina.">Adeno-associated viral vectors and the retina.</a> Adv Exp Med Biol. 613, 121-128 (2008).</li><li>Puche, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+environmental+factors+associated+with+reticular+pseudodrusen+in+age-related+macular+degeneration.">Genetic and environmental factors associated with reticular pseudodrusen in age-related macular degeneration.</a> Retina. 33 (5), 998-1004 (2013).</li><li>Campochiaro, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+for+neovascular+age-related+macular+degeneration.">Gene transfer for neovascular age-related macular degeneration.</a> Hum Gene Ther. 22 (5), 523-529 (2011).</li><li>Park, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+amyloid+beta+alters+the+tight+junction+of+retinal+pigment+epithelium+in+5XFAD+mice.">Intracellular amyloid beta alters the tight junction of retinal pigment epithelium in 5XFAD mice.</a> Neurobiol Aging. 35 (9), 2013-2020 (2014).</li><li>Shin, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+cleavage+of+amyloid+beta+by+a+viral+protease+NIa+prevents+amyloid+beta-mediated+cytotoxicity.">Intracellular cleavage of amyloid beta by a viral protease NIa prevents amyloid beta-mediated cytotoxicity.</a> PLoS One. 9 (6), e98650 (2014).</li></ol>

The authors have nothing to disclose.

이 비디오 문서에서는, 우리는 RPE / 맥락막 / 공막 평면 마운트의 대표적인 결과를 상세하게 윤부-접근 망막 주입 기술을 설명했다. 이것은 망막 색소 상피에 바이러스 벡터의 망막 주입을위한 쉽고 편리한 기술이다. 주입시 여과포 형성의 직접 시각화는 초보자를위한 정확한 전달을위한 중요한 단계입니다. 시각 실험 8-10의 저널에 소개 된 일부 망막 사출 기술이있다. 망막 하 공간이 망?...

안과에서, 유전자 치료는 단일 유전자 상속 망막증의 치료법으로서 등장했다. Leber 씨 선천성 amurosis 1,2, 색소 성 망막염 (3), (4)를 포함 choroideremia 망막 색소 상피 (RPE) 유전자와 연관된 상속 망막 병증이있다. 유전자 치료의 연구 분야는 전임상 연구 및 아데노 -associated 바이러스 (AAV), 렌티 바이러스 (LV) 및 아데노 바이러스 (AD)와 같은 바이러스 벡터를 사용하여 임상 시험 모두에서 확대되고있다 (5). 다른 바이러스 성 벡터는 망막에 다른 친 화성이있다. 안전하고 효과적인 유전자 치료의 경우, 바이러스 벡터 신중 표적 세포와 표적 유전자에 따라 선택되어야한다. 효율적인 유전자 전달이 표적 세포에 대한 유전자 전달의 경로는 따라서 신중뿐만 선택되어야한다, 또한 중요하다. 바이러스 벡터의 인공 전달을위한 가장 일반적인 두 가지 방법이 subret된다원고 판 주입과 유리체 강내 주입 6. 후자 주입술 널리 당뇨 망막 7 습식 노인성 황반변 성 (AMD) 및 황반부 종의 맥락막 혈관 신생을 치료하는 약물 전달을 위해 사용되었다. 유리체 강내 경로 및 내측 망막 바이러스 벡터의 노광을 제공하지만, 외측 망막 벡터의 확산이 제한된다. 한편, 망막 경로 지역화 여과포를 유도, 망막 및 RPE 간의 잠재적 바이러스 벡터 공간으로의 직접 전달을 제공한다. 따라서, 망막 하 주사 현재 시각 세포 및 RPE를 타겟팅보다 효율적인 경로로 간주된다. 수술 방법의 측면에서, 인간 환자 유리체 망막 손상을 방지하기 위해 주입술 안전한 영역으로서 선택된다 갈 거예요. 단순히 쥐에이 방법을 수정하여, 우리는 윤부 접근 방식을 통해 subretinally 또는 intravireally 바이러스 벡터를 주입 할 수있다. 이 비디오에서기사, 우리는 쥐의 망막 색소 상피에 바이러스 벡터의 망막 주사의 쉽고 편리한 방법을 보여줍니다. 30 G 1/2 바늘 윤부에 후방에서 단일 천자 후, 33 G 무딘 바늘 장착 된 마이크로 리터 주사기는 윤부 천자 사이트를 통해 망막 하 공간에 삽입됩니다. 1.5의 바이러스 성 벡터 - 2 μL 볼륨은 망막과 망막 색소 상피가 망막 소포를 유도 사이의 잠재적 인 공간으로 주입된다. 이 절차는 수술 현미경을 사용하는 직접적인 시각화 하에서 수행 될 수있다. 반복 연습도 여과포 형성의 직접 시각화없이 복제 결과를 보장합니다. 이 쥐의 망막 색소 상피에서 망막 유전자 전달에 대한 정확하고 시간을 절약 실험을 수행하는 연구자 도움이 될 것입니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>TWEEZERS DUMONT #5 11cm DUMOSTAR 0.1 x 0.06 mm TIPS</td>
			<td>WPI</td>
			<td>500233</td>
			<td></td>
		</tr>
		<tr>
			<td>VANNAS Scissors S/S, 105mm</td>
			<td>WPI</td>
			<td>555583S</td>
			<td></td>
		</tr>
		<tr>
			<td>33G Blunt needle</td>
			<td>WPI</td>
			<td>NF33BL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoFil Syringe, 10 microliter&#160;</td>
			<td>WPI</td>
			<td>NANOFIL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPE-KIT</td>
			<td>WPI</td>
			<td>RPE-KIT</td>
			<td>For easy one hand injection</td>
		</tr>
		<tr>
			<td>30Gx1/2 (0.3mmx 13mm) BD PrecisionGlideTM Needle</td>
			<td>BD</td>
			<td>305107</td>
			<td>Initial puncture for subretinal injection</td>
		</tr>
		<tr>
			<td>Microscope Cover Glasses (No. 1 3 mm diameter)</td>
			<td>Warner Instruments</td>
			<td>64-0720&#160; (CS-3R)</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica operating microscope</td>
			<td>Leica</td>
			<td>LM M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresecein microscope</td>
			<td>Nikon</td>
			<td>Eclipse 80i</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentivirus</td>
			<td>Thermo scientific</td>
			<td>TMO.LV-Ctr</td>
			<td>Used to dilute vectors</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-015</td>
			<td>Used to dilute vectors</td>
		</tr>
		<tr>
			<td>Troperin (Phenylephrin 0.5%-Tropicamide 0.5%)</td>
			<td>Hanmi</td>
			<td></td>
			<td>For dilation</td>
		</tr>
		<tr>
			<td>Proparacaine Hydrochloride Ophthalmic Solution USP, 0.5% (Sterile)</td>
			<td>Bausch&#38;Lomb</td>
			<td></td>
			<td>For topical anesthesia</td>
		</tr>
		<tr>
			<td>Healon GV OVD</td>
			<td>Abbott Medical Optics Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil 50 (tiletamine hypochloride and zolazepam hypochloride)</td>
			<td>Virbac</td>
			<td></td>
			<td>For general anesthesia</td>
		</tr>
		<tr>
			<td>Rompun&#174; injection (Xylazine HCl)</td>
			<td>Bayer</td>
			<td></td>
			<td>For general anesthesia</td>
		</tr>
	</tbody>
</table>

limbal approach-subretinal injection of viral vectors for gene therapy in mice retinal pigment epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

이 프로토콜에 의해 바이러스 성 유전자 전달에 망막 주사의 효능을 평가하기 위해, 우리는 인디케이터 GFP 및 RFP를 모두 표현하는 CMV 프로모터와 시판 LV 벡터를 사용했다. 눈은 연구 목적에 따라 적절한 시간 후에 적출 하였다. 대표적인 결과를 들어, 눈을 10 주에서 망막 주사 후 20 주 적출 하였다. 전술 한 방법을 사용하여 망막의 완전한 제거 후, ​​RPE / 맥락막 / 공막 복합체의 평면 실장 형광 ...

Watch this Scientific Journal Video about Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium at JoVE.com

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

마우스 망막 색소 상피의 유전자 치료에 대한 바이러스 벡터의 윤부 접근 - 망막 주입

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

limbal-approach-subretinal-injection-viral-vectors-for-gene-therapy

Research

JoVE Journal

Neuroscience

21.0K 조회수.  Seoul National University College of Medicine. Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

비디오: Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

망막 안료 상피 생리학과 Pathophysiology 연구를위한 실험 모델

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

연구

신경과학

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an injecting needle produces during its trajectory is a major concern especially in the mouse brain for not only the brain is small but also sometimes multiple injections are needed.  We show here a method to produce glass capillary needles with a 50-&mu;m lumen which significantly reduces the brain damage and allows a precise targeting into the rodent brain. This method allows a delivery of small volumes (from 20 to 100 nl), reduces bleeding risks, and minimizes passive diffusion of drugs into the brain parenchyma. By using different size of capillary glass tubes, or changing the needle lumen, several types of substances and cells can be injected. Microinjections with a glass capillary tube represent a significant improvement in injection techniques and deep brain targeting with minimal collateral damage in small rodents.

In this article, we show a method to make glass capillary needles with a 50-&mu;m lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain.

마약, 바이러스성 벡터, 또는 세포 이식 전달 Microinjections 깊은 뇌 구조의 타겟팅

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Adeno - 관련 바이러스성 벡터의 Intracranial 분사

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

망막 안료 상피의 전체 마운트를위한 마우스 아이의 해부

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods 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.

의 배아와 성인 신경 줄기 세포의 확장<em&gt; 자궁에</em&gt; Electroporation 또는 바이러스 성 Stereotaxic 주입

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

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

마우스 척추 조건부 유전자 조작을위한 바이러스 성 벡터의 Stereotaxic 주입

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

영구와 광범위한 신경 세포의 형질 도입에 대한 신생아 마우스 뇌의 뇌실 바이러스 감염

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

쥐의 눈으로 인간 망막 안료 상피 세포의 트랜스 scleral Subretinal 이식에 대 한 세련 된 프로토콜의 개발

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

마우스에 Transcranial Photobiomodulation 치료에 대 한 프로토콜

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

바이러스 벡터의 intracerebral 주입에 의해 인터뉴런으로 상주 신경교 세포의 생체 내 직접 재프로그래밍

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...