이 작품은 국립 자연 과학 재단의 중국 (보조금 번호 81570912, 81771016, 81100717)에 의해 지원 되었다.

모든 절차와 동물 수술 동물 관리 및 사용 위원회 중국의 자본 의료 대학의의 지침에 따라 실시 했다.
1. 장치 준비
<ol><li>주입 정 (그림 1A)을 하려면 폴 리 이미 드 튜브를 연결 (내부 직경 114.3 µ m, 외경 139.7 µ m, 길이 3 cm) 폴 리 에틸렌 튜브를 (내부 직경 280 μ m, 외경 640 µ m, 길이 40 cm). Superglue를 사용 하 여 적어도 3 개의 응용 프로그램과 함께 연결 인감. 에틸렌 산화물과 주입 정 소독. 참고: 배관 밀봉 할 경우는 정 맥의 방해 귀 착될 수 있다 폴 리 이미 드 튜브에 들어가기에서 superglue를 방지 합니다. 가스 살 균 연결에서 누설 될 수 있습니다 때문에 적어도 세 번에 튜브를 봉인.</li>
	<li>30g 바늘을 사용 하 여, 정상적인 염 분을 포함 하는 1 cc 주사기를 폴 리 에틸렌 튜브의 끝을 연결 합니다. 어떤 누설 또는 폴 리 이미 드 튜브와 폴 리 에틸렌 튜브 사이의 연결에 막힘 확인 1 cc 주사기로 주입 하 여 일반적인 염 분으로는 정을 채우십시오. 참고: 지금까지 누설 또는 연결의 막힌 경우에은 정 다음 절차에서 사용할 수 없습니다.</li>
	<li>일반적인 염 분으로 10 µ L 마이크로 주사기를 철수 하 고 주입 정와 상기 30 G 바늘에 연결. Microinjection 펌프 (그림 1B)에 마이크로 주사기를 설치 합니다. 0.5 µ L/min를 주입 속도 빠른 녹색 염료 및 AAV8-GFP, 1 µ L 볼륨 또는 2 µ L 스에 대 한 설정 합니다. 참고: 사출 속도의 권장된 범위는 0.1−0.5 µ L/min, 그리고 주입 볼륨의 권장된 범위는 0.5−2 µ L.</li>
	<li>1 µ L를 마이크로 주사기를 철수 하 고 주입 시 약을 추출 합니다. 일반적인 염 분 및 주입된 시 약 (그림 1C) 사이에 어 갭 형성할 것 이다.</li>
	<li>필요한 경우 마커 펜을 사용 하 여 폴 리 에틸렌 배관에 가스-액체 테두리의 위치를 레이블을 지정 합니다. 이 주입 중 시 약 흐름을 모니터링 하는 데 사용할 수 있습니다.</li>
</ol>2입니다. 성인 쥐에 Canalostomy
<ol><li>성인 마우스 anesthetize (여성, FVB/N, 5에 6 주 된) 케 타 민 HCl (120 mg/kg)의 복 주입 및 xylazine HCl (7 mg/kg). 동물 전시 고통 스러운 자극 (발가락 핀치 반사)에 대 한 응답이 될 때까지 5−10 분을 기다립니다. 마 취 후 따뜻한 전기 패드에 동물을 놓습니다. 진통제, Meloxicam (1 mg/kg), 수술 전에 피하 적용 되었습니다.</li>
	<li>동물의 눈 눈 연 고와 커버. 전기 동물 깎기로 왼쪽된 후 귀의 영역을 면도 하 고 세 번 75% 에탄올과 피부를 소독.</li>
	<li>데워 전기 패드에 동물을 놓습니다. ~ 37 ° c.에 전기 패드의 온도 설정 왼쪽된 귀에 수술을 용이 하 게 하는 오른쪽 측면 위치에 동물을 배치 합니다.</li>
	<li>1-1.5 cm 후 귀의 절 개 ~ 3mm 왼쪽된 retroauricular 홈 (그림 2A)에서 확인 합니다.</li>
	<li>후부 반원의 운하 (PSC)와 측면 반원형 운하 (LSC) 위치: pinna (굵게 도트 그림2b에서)의 루트 3 9 시로 기원과는 calvarium에 평행한 평면으로 정의 됩니다, PSC와는 LSC는 일반적으로 3 m m 2와 3 시 (그림 2B) 사이의 pinna의 루트에서에 위치 해 있습니다.</li>
	<li>퉁 명 스럽게 근육 측 뼈 PSC 및 LSC, 누구의 여백은 측 뼈 (그림 2C)에 어두운 줄무늬도 명확 하 게 보이는 노출 마이크로 포 셉와 함께 취재를 해 부. LSC에는 한 약 30 °, calvarium에 평행한 평면에서 각도 PSC는 LSC (그림 2C)에 수직. 마이크로-집게와 근육의 작은 조각을 수집 하 고 말리 면. 다음 단계에서이 근육 구멍을 밀봉 하기 위하여 사용 됩니다. 참고: 과도 한 근육에 상해를 최소화 하기 위해 조직의 찢 어 하지 마십시오. PSC (그림 2D) 노출 때 인접 한 배를 손상 하지 마십시오.</li>
	<li>26 G 바늘 (그림 2D)를 사용 하 여 PSC의 중간 부분에 작은 구멍을 확인 합니다. 구멍을 통해 유체 누설 PSC의 뼈 벽의 성공적인 침투를 나타냅니다. 적절 한 크기로, 폴 리 이미 드 튜브의 직경 보다 약간 큰 구멍을 확대. 참고: 드릴 구멍을 부드럽게 하 고 점차적으로 확대 및 PSC의 파괴를 피하기 위해.</li>
	<li>코 튼 펠 렛을 사용 하 여 PSC의 구멍을 둘러싼 유출 청소.</li>
	<li>1−2 m m (그림 2E)의 깊이에 cru 코뮌으로 PSC에 폴 리 이미 드 튜브의 끝을 부드럽게 삽입 합니다. 주입 펌프에 '실행' 버튼을 눌러 시작 합니다.</li>
	<li>주입, 후 확산 시 있도록 ~ 2 분 기다립니다. 2.6 마이크로 위 단계에서 수집 된 근육의 작은 조각을 잘라. 다음, 주입 정 맥을 제거 하 고 즉시 PSC에 있는 구멍에 근육을 배치. 참고: 새는 구멍에서 구멍 완전히 밀폐 되도록 연결 후 모든 액체에 대 한 확인 하십시오.</li>
	<li>분리 된 근육과 피하 조직 반환. 5-0 봉합 사를 사용 하 여 절 개 봉합 절 개 지역 povidone 요오드 소독. 참고: 위의 모든 수술 절차는 약 25 분 소요.</li>
	<li>위치는 전기 패드에 동물 ~ 37 ° c.에 preheated 복구에 대 한 오른쪽 측면 위치에 동물을 배치 합니다.</li>
	<li>청각 brainstem 응답 (ABR) 측정을 수행 하 고 테스트 1 주일 후 수술18수영.</li>
	<li>필요한 경우, PSC 또는 LSC의 다른 영역에 반복된 주사를 수행 합니다.</li>
</ol>3입니다. 신생아 쥐에 Canalostomy
<ol><li>저체온증을 사용 하 여 유도 하 고 출생 후 일 2 1 (P1-2) 신생아 쥐 (FVB/N)에 진정을 유지. ~ 4 분 동안 얼음의 플라스틱 랩 덮여 침대에 강아지를 두십시오. 다음, 얼음 가득 플랫폼에 강아지를 하다. 75% 에탄올과 세 번 수술 필드를 소독. 참고: 강아지의 머리는 얼음에서 인지 확인 합니다. 얼음 가득한 플랫폼에 강아지와 함께 전체 수술을 수행 합니다.</li>
	<li>다음 단계는 성인 쥐에 비해 신생아에서 다르다.<ol>
<li>~ 2 m m에서 ~ 3mm-postauricular 절 개 부위 주름에 후부 확인 (그림 3A -B).</li>
		<li>부드럽게 26 G 바늘을 사용 하 여 부드러운 PSC에 개방을 확인 합니다. 개방을 확대 하지 않고 PSC는 캐 뉼 러 삽입 (그림 3C -E).</li>
		<li>주입 후 근육의 조각을 사용 하 여 보다는 커버, 플러그, 오프닝으로 후자는 부드러운 PSC (그림 3 층)의 골절으로 이어질 수 있습니다.</li>
		<li>6-0 봉합 사를 사용 하 여 피부를 닫습니다.</li>
		<li>ABR 측정을 수행 하 고 테스트 P3030에서 수영.</li>
	</ol>
</li>
	<li>부모의 인 육은 신생아 수술 후 일반적인 문제 이다. 다음 단계는 부모의 식인 풍습의 가능성을 줄일 수 있습니다.<ol>
<li>혈액 알코올 잎사귀 절 개 수술 후 주변 정리.</li>
		<li>확인을 반환 하기 전에 신생아가 자유롭게 이동할 수 있습니다.</li>
		<li>어머니의 감 금 소, 장소 쓰레기 가운데 다시는 신생아에서 더럽혀진된 침구와 신생아를 얼룩.</li>
		<li>수술 하지 않는 어떤 유사한 postauricular 절 개 및 봉합을 받아야 한다.</li>
		<li>사육 장에서 남성을 구분 합니다.</li>
	</ol>
</li>
</ol>

<ol>
	<li>Stachler, R. 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=Clinical+practice+guideline:+sudden+hearing+loss.">Clinical practice guideline: sudden hearing loss.</a> Otolaryngol Head Neck Surg. 146 (3 Suppl), S1-S35 (2012).</li><li>Lentz, J. 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=Rescue+of+hearing+and+vestibular+function+by+antisense+oligonucleotides+in+a+mouse+model+of+human+deafness.">Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness.</a> Nat Med. 19 (3), 345-350 (2013).</li><li>Rivera, T., Sanz, L., Camarero, G., Varela-Nieto, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+delivery+to+the+inner+ear:+strategies+and+their+therapeutic+implications+for+sensorineural+hearing+loss.">Drug delivery to the inner ear: strategies and their therapeutic implications for sensorineural hearing loss.</a> Curr Drug Deliv. 9 (3), 231-242 (2012).</li><li>El Kechai, 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=Recent+advances+in+local+drug+delivery+to+the+inner+ear.">Recent advances in local drug delivery to the inner ear.</a> Int J Pharm. 494 (1), 83-101 (2015).</li><li>Akil, O., Rouse, S. L., Chan, D. K., Lustig, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+method+for+virally+mediated+gene+delivery+to+the+mouse+inner+ear+through+the+round+window+membrane.">Surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane.</a> J Vis Exp. (97), e52187 (2015).</li><li>Ahmed, H., Shubina-Oleinik, O., Holt, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+Gene+Therapies+for+Genetic+Hearing+Loss.">Emerging Gene Therapies for Genetic Hearing Loss.</a> J Assoc Res Otolaryngol. 18 (5), 649-670 (2017).</li><li>Murillo-Cuesta, S., 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=A+Comparative+Study+of+Drug+Delivery+Methods+Targeted+to+the+Mouse+Inner+Ear:+Bullostomy+Versus+Transtympanic+Injection.">A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection.</a> J Vis Exp. (121), e54951 (2017).</li><li>Stevens, S. M., Brown, L. N., Ezell, P. C., Lang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mouse+Round-window+Approach+for+Ototoxic+Agent+Delivery:+A+Rapid+and+Reliable+Technique+for+Inducing+Cochlear+Cell+Degeneration.">The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration.</a> J Vis Exp. (105), e53131 (2015).</li><li>Salt, A. N., Plontke, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+local+drug+delivery+to+the+inner+ear.">Principles of local drug delivery to the inner ear.</a> Audiol Neurootol. 14 (6), 350-360 (2009).</li><li>Akil, O., 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+hearing+in+the+VGLUT3+knockout+mouse+using+virally+mediated+gene+therapy.">Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.</a> Neuron. 75 (2), 283-293 (2012).</li><li>Pan, 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=Gene+therapy+restores+auditory+and+vestibular+function+in+a+mouse+model+of+Usher+syndrome+type+1c.">Gene therapy restores auditory and vestibular function in a mouse model of Usher syndrome type 1c.</a> Nat Biotechnol. 35 (3), 264-272 (2017).</li><li>Kilpatrick, L. A., 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=Adeno-associated+virus-mediated+gene+delivery+into+the+scala+media+of+the+normal+and+deafened+adult+mouse+ear.">Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear.</a> Gene Ther. 18 (6), 569-578 (2011).</li><li>Izumikawa, 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=Auditory+hair+cell+replacement+and+hearing+improvement+by+Atoh1+gene+therapy+in+deaf+mammals.">Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals.</a> Nat Med. 11 (3), 271-276 (2005).</li><li>Chang, Q., 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=Virally+mediated+Kcnq1+gene+replacement+therapy+in+the+immature+scala+media+restores+hearing+in+a+mouse+model+of+human+Jervell+and+Lange-Nielsen+deafness+syndrome.">Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome.</a> EMBO Mol Med. 7 (8), 1077-1086 (2015).</li><li>Chen, Z., Mikulec, A. A., McKenna, M. J., Sewell, W. F., Kujawa, S. 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+method+for+intracochlear+drug+delivery+in+the+mouse.">A method for intracochlear drug delivery in the mouse.</a> J Neurosci Methods. 150 (1), 67-73 (2006).</li><li>Kawamoto, K., Oh, S. H., Kanzaki, S., Brown, N., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+and+structural+outcome+of+inner+ear+gene+transfer+via+the+vestibular+and+cochlear+fluids+in+mice.">The functional and structural outcome of inner ear gene transfer via the vestibular and cochlear fluids in mice.</a> Mol Ther. 4 (6), 575-585 (2001).</li><li>Bowers, W. 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=Neurotrophin-3+transduction+attenuates+cisplatin+spiral+ganglion+neuron+ototoxicity+in+the+cochlea.">Neurotrophin-3 transduction attenuates cisplatin spiral ganglion neuron ototoxicity in the cochlea.</a> Mol Ther. 6 (1), 12-18 (2002).</li><li>Wang, G. P., 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=Adeno-associated+virus-mediated+gene+transfer+targeting+normal+and+traumatized+mouse+utricle.">Adeno-associated virus-mediated gene transfer targeting normal and traumatized mouse utricle.</a> Gene Ther. 21 (11), 958-966 (2014).</li><li>Isgrig, 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=Therapy+Restores+Balance+and+Auditory+Functions+in+a+Mouse+Model+of+Usher+Syndrome.">Therapy Restores Balance and Auditory Functions in a Mouse Model of Usher Syndrome.</a> Mol Ther. 25 (3), 780-791 (2017).</li><li>Gassner, D., Durham, D., Pfannenstiel, S. C., Brough, D. E., Staecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canalostomy+as+a+surgical+approach+for+cochlear+gene+therapy+in+the+rat.">Canalostomy as a surgical approach for cochlear gene therapy in the rat.</a> Anat Rec (Hoboken). 295 (11), 1830-1836 (2012).</li><li>Yamasoba, T., Yagi, M., Roessler, B. J., Miller, J. M., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inner+ear+transgene+expression+after+adenoviral+vector+inoculation+in+the+endolymphatic+sac.">Inner ear transgene expression after adenoviral vector inoculation in the endolymphatic sac.</a> Hum Gene Ther. 10 (5), 769-774 (1999).</li><li>Xia, L., Yin, S., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inner+ear+gene+transfection+in+neonatal+mice+using+adeno-associated+viral+vector:+a+comparison+of+two+approaches.">Inner ear gene transfection in neonatal mice using adeno-associated viral vector: a comparison of two approaches.</a> PLoS One. 7 (8), e43218 (2012).</li><li>Chien, W. W., McDougald, D. S., Roy, S., Fitzgerald, T. S., Cunningham, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+gene+transfer+mediated+by+adeno-associated+virus:+Comparison+of+two+surgical+approaches.">Cochlear gene transfer mediated by adeno-associated virus: Comparison of two surgical approaches.</a> Laryngoscope. 125 (11), 2557-2564 (2015).</li><li>Zhu, B. Z., Saleh, J., Isgrig, K. T., Cunningham, L. L., Chien, 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=Hearing+Loss+after+Round+Window+Surgery+in+Mice+Is+due+to+Middle+Ear+Effusion.">Hearing Loss after Round Window Surgery in Mice Is due to Middle Ear Effusion.</a> Audiol Neurootol. 21 (6), 356-364 (2017).</li><li>Wang, Y., 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=Early+postnatal+virus+inoculation+into+the+scala+media+achieved+extensive+expression+of+exogenous+green+fluorescent+protein+in+the+inner+ear+and+preserved+auditory+brainstem+response+thresholds.">Early postnatal virus inoculation into the scala media achieved extensive expression of exogenous green fluorescent protein in the inner ear and preserved auditory brainstem response thresholds.</a> J Gene Med. 15 (3-4), 123-133 (2013).</li><li>Lee, M. Y., 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=Survival+of+human+embryonic+stem+cells+implanted+in+the+guinea+pig+auditory+epithelium.">Survival of human embryonic stem cells implanted in the guinea pig auditory epithelium.</a> Sci Rep. 7, 46058 (2017).</li><li>Ishimoto, S., Kawamoto, K., Kanzaki, S., Raphael, Y. <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+into+supporting+cells+of+the+organ+of+Corti.">Gene transfer into supporting cells of the organ of Corti.</a> Hear Res. 173 (1-2), 187-197 (2002).</li><li>Okada, H., 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+transfer+targeting+mouse+vestibule+using+adenovirus+and+adeno-associated+virus+vectors.">Gene transfer targeting mouse vestibule using adenovirus and adeno-associated virus vectors.</a> Otol Neurotol. 33 (4), 655-659 (2012).</li><li>Suzuki, J., Hashimoto, K., Xiao, R., Vandenberghe, L. H., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+gene+therapy+with+ancestral+AAV+in+adult+mice:+complete+transduction+of+inner+hair+cells+without+cochlear+dysfunction.">Cochlear gene therapy with ancestral AAV in adult mice: complete transduction of inner hair cells without cochlear dysfunction.</a> Sci Rep. 7, 45524 (2017).</li><li>Guo, J. Y., 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=Cochleovestibular+gene+transfer+in+neonatal+mice+by+canalostomy.">Cochleovestibular gene transfer in neonatal mice by canalostomy.</a> Neuroreport. 28 (11), 682-688 (2017).</li><li>Beyea, J. A., Agrawal, S. K., Parnes, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmastoid+semicircular+canal+occlusion:+a+safe+and+highly+effective+treatment+for+benign+paroxysmal+positional+vertigo+and+superior+canal+dehiscence.">Transmastoid semicircular canal occlusion: a safe and highly effective treatment for benign paroxysmal positional vertigo and superior canal dehiscence.</a> Laryngoscope. 122 (8), 1862-1866 (2012).</li><li>Naples, J. G., Eisen, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+History+and+Evolution+of+Surgery+on+the+Vestibular+Labyrinth.">The History and Evolution of Surgery on the Vestibular Labyrinth.</a> Otolaryngol Head Neck Surg. 155 (5), 816-819 (2016).</li><li>Hamilton, L., Keh, S., Spielmann, P. M., Hussain, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+we+do+it:+locating+the+posterior+semicircular+canal+in+occlusion+surgery+for+refractory+benign+paroxysmal+positional+vertigo:+a+cadaveric+temporal+bone+study.">How we do it: locating the posterior semicircular canal in occlusion surgery for refractory benign paroxysmal positional vertigo: a cadaveric temporal bone study.</a> Clinical Otolaryngology. 41 (2), 190-193 (2016).</li><li>Jung, J. Y., 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=siRNA+targeting+Hes5+augments+hair+cell+regeneration+in+aminoglycoside-damaged+mouse+utricle.">siRNA targeting Hes5 augments hair cell regeneration in aminoglycoside-damaged mouse utricle.</a> Mol Ther. 21 (4), 834-841 (2013).</li><li>Kamiya, 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=Mesenchymal+stem+cell+transplantation+accelerates+hearing+recovery+through+the+repair+of+injured+cochlear+fibrocytes.">Mesenchymal stem cell transplantation accelerates hearing recovery through the repair of injured cochlear fibrocytes.</a> Am J Pathol. 171 (1), 214-226 (2007).</li><li>Pfannenstiel, S. C., Praetorius, M., Plinkert, P. K., Brough, D. E., Staecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-2+gene+therapy+prevents+aminoglycoside-induced+degeneration+of+auditory+and+vestibular+hair+cells.">Bcl-2 gene therapy prevents aminoglycoside-induced degeneration of auditory and vestibular hair cells.</a> Audiol Neurootol. 14 (4), 254-266 (2009).</li><li>Kawamoto, K., Izumikawa, M., Beyer, L. A., Atkin, G. M., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+hair+cell+regeneration+in+the+mouse+utricle+following+gentamicin+ototoxicity.">Spontaneous hair cell regeneration in the mouse utricle following gentamicin ototoxicity.</a> Hear Res. 247 (1), 17-26 (2009).</li><li>Wang, G. P., 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=Notch+signaling+and+Atoh1+expression+during+hair+cell+regeneration+in+the+mouse+utricle.">Notch signaling and Atoh1 expression during hair cell regeneration in the mouse utricle.</a> Hear Res. 267 (1-2), 61-70 (2010).</li><li>Pietola, L., 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=HOX-GFP+and+WOX-GFP+lentivirus+vectors+for+inner+ear+gene+transfer.">HOX-GFP and WOX-GFP lentivirus vectors for inner ear gene transfer.</a> Acta Otolaryngol. 128 (6), 613-620 (2008).</li><li>Han, J. 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=Transgene+expression+in+the+guinea+pig+cochlea+mediated+by+a+lentivirus-derived+gene+transfer+vector.">Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector.</a> Hum Gene Ther. 10 (11), 1867-1873 (1999).</li></ol>

관심 없음 충돌 선언 됩니다.

이 연구에서 우리는 canalostomy에 의해 약물 전달 달팽이 관 vestibular 끝-장기에 걸쳐 시 약의 광범위 한 배포 결과 다는 것을 보였다. 내가 유전자 전달 방법으로 canalostomy는 청각 및 vestibular 함수에 최소한의 피해와 성인 및 신생아 쥐의 내 귀에 GFP 식에서 결과. 또한, 여러 주사 같은 동물에서 쉽게 수행할 수 있습니다.
그것은 특히 성인 쥐16,<sup class="xre...

감각 청력 손실 및 영향을 vestibular 부전 환자와의 밀접 하 게 연관 된 내가 장애. 내가 치료 약물의 배달 내가 질환의 치료에 대 한 약속을 보여줍니다. 조직 또는 로컬 접근 내가 약물을 전달 하기 위해 사용할 수 있습니다. 일부 내가 질병 idiopathic 갑자기 청력 상실, 일반적으로 전신 스테로이드1처리 등 조직의 약국 성공적으로 처리 됩니다. 또한, Lentz 외. 센스 oligonucleotide의 체계적 관리는 난청을 개선 하 고 Ush1c 돌연변이 마우스 모델2에서 기능을 균형 있게 보여주었다. 그러나, 내가 질환의 많은 부분이 처리 되지 않습니다 효과적으로 조직의 의약품 안전 청에 의해 내가3,4에 마약 사용을 제한 하는 혈액-미로 장벽 때문에. 반면, 지역 마약 배달 전략 내가 장애를 보다 효율적으로 처리할 수 있습니다. 사실, 내가 잠재적으로 로컬 약물 전달;에 대 한 이상적인 목표 한 사이트 유포 또는 주입 후 약의 보급을 용이 하 게, 유체로 가득 그리고 인접 장기에서 상대적으로 고립 된 부작용5,6제한.
지역 마약 배달 전략 intratympanic 및 intralabyrinthine 메서드가 포함 됩니다. Intratympanic 루트의 효과 크게 둥근 창 막 (RWM)와 RWM3,4,,78에 약물의 체류 시간을 통해 마약 침투성에 의존합니다. 따라서, 약물 이나는 RWM 뚫을 수 없는 시 약의 납품에 대 한 적당 하다. Intralabyrinthine 메서드는 약물의 높은 복용량에 광범위 하 게 배포 결과 내가 직접 접종을 포함 한다. 그러나, intralabyrinthine 방법 섬세 한 수술을 요구 하 고 침략 적, 선도 내가 기능을 손상 하는. 현재, 인간9에 사용 하기 위해 충분히 안전한 것으로 증명 되지 않았습니다 intralabyrinthine 주입 약물의 동물 연구에만 사용 됩니다. 따라서, 수술 절차를 단순화 해야 합니다, 그리고 상해의 위험 감소 병원에 intralabyrinthine 접근을 번역 하.
몇몇 intralabyrinthine 접근 RWM5,,1011 와 스칼라 미디어12,,1314, scala tympani에 주입 하 여 동물에 부과 하 고 있다 15 , 16, 스칼라 현관17, 반원의 운하16,18,,1920그리고21endolymphatic sac. 방법 마다 장단점6. 납품은 RWM 통해 신생아 쥐5,22에 atraumatic입니다. 그러나, 가벼운 난청 RWM 주입23, 수술24후 중이 토로 인해 가능성이 후 성인 쥐에서 관찰 됩니다. 대상 최종 기관12,14, 에서 높은 시 약 농도 달성 하는 스칼라 미디어 주입, 직접 우주로 endolymphatic 감각 상피를 포함 하는 시 약의 주입을 포함 한다, 25 , 그러나 26.,이 이렇게 복잡 한 절차를 요구 한다 고 심리 임계값의 상당한 상승 산 후 날 5 (P5) 보다 수행 하는 경우25,27, 그것의 응용 프로그램을 제한.
위에서 언급 한 intralabyrinthine 방식에 비해, canalostomy 특히 성인 쥐16,18,,2829,30에서 내가 최소한의 손상을 일으키는합니다 보호 효과 및 변환 측면의 평가 대 한 중요 한. 또한, 설치류, 반원의 운하 넘어 물집, 수술 절차를 용이 하 게 하 고 수술 하는 동안 중이 장애를 피할 수 있습니다. 클리닉, 반원의 운하 수술 다루기 힘든 양성 발작의 자세 현기증31,,3233, canalostomy의 임상 타당성을 제안에 대 한 사용 됩니다. 이후 처음가 모토 외설명. 16 , 2001 년에 canalostomy murine 내가18,,1928,29로 바이러스 성 벡터, siRNA, 줄기 세포, aminoglycoside, 등 다양 한 시 약을 제공 하는 데 사용 되었습니다. ,,3435,,3637. Canalostomy에 의해 adeno 관련 바이러스 (AAV) 벡터의 접종 가능 감각 상피에 외 인 유전자의 overexpression 달팽이 관 및 vestibular 끝-장기18,28, 의 기본 신경 29,30. Canalostomy에 의해 유전자 치료를 whirlin 균형 기능을 복원 하 고 인간의 안내인 증후군19, 그 canalostomy 유전자 cochleovestibular 질병에 대 한 유전자 치료의 연구에 대 한 유용한 제안 마우스 모델에서 청력을 향상 시킵니다. Canalostomy에 의해 중간 엽 줄기 세포의 이식 결과 인공 fibrocytes와 급성 감각 청각 손실35의 쥐 모델에서 청력 회복의 개편. 또한, canalostomy aminoglycosides vestibular 병 변18,,3438, 확립 하 내가 사용할 수 있습니다. 그리고18 필요한 경우 여러 주사를 수행할 수 있습니다. , 34.
현재 문서에서 우리, 자세히 설명, 성인 및 신생아 쥐에 canalostomy 기술. 우리 canalostomy 후 즉각적이 고 장기 결과 평가 하기 위해 마우스 내가 녹색 형광 단백질 (GFP) 유전자 (AAV8-GFP)와 스, 빠른 녹색 염료 및 AAV serotype 8 (AAV8)를 포함 하 여 다양 한 시 약을 주사.

<table>
	<tbody>
		<tr>
			<td>Polymide Tubing</td>
			<td>A-M Systems</td>
			<td>823400</td>
			<td></td>
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			<td>Polyethylene Tubing</td>
			<td>Scientific Commodities Inc.</td>
			<td>BB31695-PE/1</td>
			<td></td>
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			<td>10&#956;l Microsyringe</td>
			<td>Hamilton Company</td>
			<td>80001</td>
			<td></td>
		</tr>
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			<td>Xylazine HCL</td>
			<td>Sigma-Aldrich Co. Llc.</td>
			<td>X-1251</td>
			<td></td>
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			<td>Operating Miroscope</td>
			<td>Carl Zeiss Optical LLC.</td>
			<td>Pico</td>
			<td></td>
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			<td>Micro Forceps</td>
			<td>Dumont Dumostar</td>
			<td>10576</td>
			<td></td>
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			<td>Fast-green Dye</td>
			<td>Sigma-Aldrich Co. Llc.</td>
			<td>F7252</td>
			<td></td>
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			<td>AAV8-GFP</td>
			<td>BioMiao Biological Technology Co. Ltd (Beijing, China)</td>
			<td>20161101</td>
			<td>Titer: 2&#215;10e12 vg/mL</td>
		</tr>
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			<td>Streptomycin Sulfate</td>
			<td>Sigma-Aldrich Co. Llc.</td>
			<td>S9137</td>
			<td></td>
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			<td>Microinjection Pump</td>
			<td>Stoelting Co.</td>
			<td>789100S</td>
			<td></td>
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			<td>Electric Pad</td>
			<td>Pet Fun</td>
			<td>11072931136</td>
			<td></td>
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			<td>1 cc Syringe</td>
			<td>Mishawa Medical Industries Ltd. (Shanghai, China)</td>
			<td>2011-3151258</td>
			<td></td>
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			<td>Ketamine HCL</td>
			<td>Gutian Pharmaceutical Co., Ltd. (Fujian, China)</td>
			<td>H35020148</td>
			<td></td>
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			<td>Electric Animal Clipper</td>
			<td>Codos Electrical Appliances Co., Ltd. (Guangdong, China)</td>
			<td>CP-8000</td>
			<td></td>
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			<td>Cotton Pellet</td>
			<td>Yatai Healthcare Ltd. (Henan, China)</td>
			<td>Yu-2008-1640081</td>
			<td></td>
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			<td>Suture</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co., Ltd. (Shanghai, China)</td>
			<td>Hu-2013-2650207</td>
			<td></td>
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			<td>Eye Ointment</td>
			<td>Beijing Shuangji Pharmaceutical Ltd. (Beijng China)</td>
			<td>H11021270</td>
			<td></td>
		</tr>
	</tbody>
</table>

canalostomy as a surgical approach to local drug delivery into the inner ears of adult and neonatal mice

내가 치료 약물의 로컬 배달 내가 질병에 대 한 유망한 요법입니다. 사출 반원의 운하 (canalostomy)를 통해 내가 지역 약물 전달에 대 한 유용한 접근 될 표시 되었습니다. 이 문서의 목표는 설명, 세부 사항, 성인 및 신생아 쥐에 있는 canalostomy에 관련 된 수술 기법입니다. 빠른 녹색 염료 및 adeno 관련 바이러스 serotype 8 녹색 형광 단백질 유전자에 의해 표시 된,는 canalostomy vestibular 끝-기관 청력에 최소한의 손상으로 달팽이 관에 삽입 된 시 약의 광범위 한 배포를 촉진 하 고 vestibular 기능입니다. 수술은 성인과 신생아 쥐;에서 성공적으로 구현 실제로, 다 수 수술 필요한 경우 수행할 수 수 있습니다. 끝으로, canalostomy 성인 및 신생아 쥐의 내 귀에 약물 전달에 효과적이 고 안전한 방법 이며 미래에 인간의 내가 질병을 치료 하는 데 사용할 수 있습니다.

빠른 녹색 염료는 내가 있는 그것의 즉각적인 배포를 평가 하는 성인 및 신생아 쥐의 PSC에 주입 했다. 염료 (그림 4) 수술 후 바로 달팽이 관, 현관, 반원의 운하에 걸쳐 발견 되었다.
안전 및 내가 유전자 납품에 대 한 canalostomy의 효율성 평가, AAV8 GFP 성인 및 신생아 쥐의 내가 주입 했다. 모든 동물 전시 정?...

Watch this Scientific Journal Video about Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice at JoVE.com

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

성인 및 신생아 쥐의 내 귀에 로컬 약물 전달에 대 한 외과 접근으로 Canalostomy

여기는 청각 및 vestibular 함수에 최소 손상 반원의 운하를 통해 성인 및 신생아 쥐의 내 귀에 로컬 약물 전달 수 있는 canalostomy 절차에 설명 합니다. 이 메서드는 마우스 내가 바이러스 성 벡터, 제약, 그리고 작은 분자를 접종을 사용할 수 있습니다.

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

canalostomy-as-surgical-approach-to-local-drug-delivery-into-inner

Research

JoVE Journal

Genetics

10.6K 조회수.  Capital Medical University. 여기는 청각 및 vestibular 함수에 최소 손상 반원의 운하를 통해 성인 및 신생아 쥐의 내 귀에 로컬 약물 전달 수 있는 canalostomy 절차에 설명 합니다. 이 메서드는 마우스 내가 바이러스 성 벡터, 제약, 그리고 작은 분자를 접종을 사용할 수 있습니다.

비디오: Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

시험 관내 전사 분석 실험 및 약물 발견에의 응용

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

연구

유전학

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

여러 형광에 의한 간 염색체 안정 수차의 검출<em&gt; 현장에서</em&gt; 하이브리드 (mFISH) 및 방사선 조사 마우스의 스펙트럼 핵형 분석 (SKY)

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

유도 성 과도 형질을 통해 제어 이온 채널 식

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

설치류에 신경 근육 학 질병을 치료 하는 재조합 형 Adeno 관련 바이러스 Serotype 9를 사용 하 여 예측에 관한 체계적 전달

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

오버랩2 방법으로 시너지 약물 조합의 높은 처리량 식별

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

복제 설정된 방법: 양적 측정 꼬마 선 충 수명에 접근 높은 처리량

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

분자 특성 조합 단일 셀 접근 및 인간의 줄기 및 조상 인구 Immunophenotypic이

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast permitting in vivo phenotypic analysis. This method does not rely on maize transformation but exploits microbial genetics and secretory capabilities of pathogens. Herein, it allows inspection of in vivo delivered secreted proteins with high spatiotemporal resolution at different kinds of infection sites and tissues. The Trojan horse strategy can be utilized to transiently complement maize loss-of-function phenotypes, to functionally characterize protein domains, to analyze off-target protein effects, or to study onside protein overdosage, making it a powerful tool for protein studies in the maize crop system. This work contains a precise protocol on how to generate a Trojan horse strain followed by standardized infection protocols to apply this method to three different maize tissue types.

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

This work describes the cloning of an Ustilago maydis Trojan horse strain for the in situ delivery of secreted maize proteins into three different types of maize tissues.

원래의 배달 옥수수 단백질에 대 한 Ustilago maydis 트로이 목마 사용 하

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

작은 분자 조사를 시험관 및 셀 모양을 사용 하 여 사전 mRNA 구조 변화 유도

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

인간으로 Epigenome 편집 도구의 효율적인 배달 lentiviral 벡터 플랫폼 유도 만능 줄기 세포 유래 질병 모델

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...