이 작품은 미국 암 학회 (American Cancer Society) 연구 학자 그랜트 (RSG-12-148-01-DDC) 및 CIRM 기본 생물학 상 (RB4-05779) supportedby했다.

인간의 피부 프로토콜 캘리포니아 대학, 샌디에고의 연구 윤리위원회의 지침에 따라 수행 하였다. 인간의 피부는 폐기 수술 샘플에서 얻은 또는 (피부 은행이 소재 / 장비의 표에 나와있는) 피부 은행에서 구입하실 수 있습니다. 피부 또는 기증자의 나이 파생 위치는 실험만큼 진피의 기저막 영역 단백질 (콜라겐 / 라미닌)에 중요하지 않은 것은 성능이 저하되지 않습니다. 약화 된 인간의 진피 1. 준비 <ol><li> 인간의 피부를 수신하면, (~ 3-5 분) 해동 조직 배양 후드에서 조직을 배치. 피부의 크기에 따라, 일회용 무균 50 ㎖ 원뿔형 튜브 또는 125 ml의 용기에 해동 조직을 배치. 피부 은행에서 피부의 전형적인 크기는 0.75 평방 피트입니다. </li><li> 1X PBS에서 4 배 페니실린과 스트렙토 마이신 (펜 / 연쇄 구균)와 75 %의 전체 컨테이너를 채 웁니다. 격렬하게 흔들어5 분 및 액체 폐기하십시오. 이 단계를 3 번​​ 이상 반복합니다. </li><li> 마지막 세척 후, 새로운 튜브 / 병에 조직을 전송하고 컨테이너의 75 %를 채우기 위해 신선한 배 펜 / 패 혈성 / PBS를 추가합니다. 2 주부터 표피 진피의 분리를 허용하기 위해 37 ℃에서 조직 배양 인큐베이터에서이 혼합물을 배양한다. </li><li> 무균 조건에서, 진피에서 표피를 벗겨 집게를 사용합니다. 표피는 기증자 (그림 1A)의 인종에 따라 색상이 반면 진피는 완전히 흰색입니다. 인체 조직을 다루는 기관의 프로토콜에 따라 표피를 폐기하십시오. </li><li> 관 또는 병에 진피를 놓고 (4 시간 5 분 세척) 활발한 진탕 1X PBS에 희석 배 펜 / 연쇄상 구균에 씻는다. 마지막 세척 후 사용할 준비가 될 때까지 4 ° C에서 4 배 펜 / 1X PBS에 희석 연쇄 구균과 상점에서 새로운 튜브 / 병에 진피를 전송합니다. 진피 년까지 4 ℃에서 저장 될 수있다. </li></OL&gt; 2. 유전자 수정 기본 인간의 각질 형성 세포 주 : 차 인간 각화 세포를 감염 바이러스를 생산하기 위해 완벽한 DMEM 배지 [DMEM + 10 % 소 태아 혈청 (FBS) + 펜 / 연쇄상] 재배 사용 amphotropic 피닉스 세포. 이러한 개그-POL 및 봉투와 같은 단백질을 인코딩 바이러스 성 포장 유전자가 안정적으로 레트로 바이러스 벡터로 형질 전환 된 경우 바이러스 생산을 허용 피닉스 세포 게놈에 통합되어 있습니다. 피닉스 세포 고효율 높은 역가의 바이러스 생산을위한 형질 허용 될 수있다. 이 포장 라인에서 생산 된 바이러스는 인간을 포함한 포유 동물 세포의 큰 다양성을 감염시킬 수 있습니다. <ol><li> 1 일 : 형질 전날, 완전한 DMEM 매체 잘 당 800,000 세포의 밀도로 6 웰 플레이트에 시드 피닉스 세포. </li><li> 주 2 : 형질의 날, 3 레트로 바이러스 벡터의 μg의 당을 잘 사용합니다. 1.5 ML 튜브에 레트로 바이러스 벡터의 나누어지는 3 μg의. 100 μ의 혼합을 사​​용하여; L의 DMEM 플러스 벡터의 모든 3 μg의에 대한 형질 전환 시약의 6 μL. 1.5 ML 튜브에 100 μL의 DMEM으로 형질 전환 시약의 6 μl를 섞는다. (사용되는 레트로 바이러스 벡터는 장비 / 재료 표에 나와 있습니다). <ol><li> 5 분 동안 인큐베이션 및 레트로 바이러스 벡터를 함유하는 3 μg의 사전 분취 튜브에 106 ㎕의 혼합물을 추가한다. 혼합하고 실온에서 30 분 동안 배양한다. 피닉스 세포의 각 웰이 전체 혼합물의 적가를 추가합니다. </li></ol></li><li> 주 3 : 형질 전환 후 날, 피닉스 세포에서 미디어를 제거하고 신선한 완전한 DMEM 배지 2 ㎖를 추가합니다. 같은 날에, 웰 당 75,000 세포의 밀도로 6 웰 플레이트에 차 인간 각화 세포를 시드. 항생제와 각질 세포의 무 혈청 배지 (KCSFM) (펜 / 연쇄상 구균)에있는 각질을 유지한다. 참고 : 1 차 인간의 각질 세포가 구입 하였다. 세포 (소재 / 장비의 표에 나와있는) 다양한 공급 업체에서 구입하실 수 있습니다. </li><li> 주 4 : 하르지금은 바이러스 입자를 포함하는 형질 전환 피닉스 세포에서 용지를 가득. 주사기를 사용하여 0.45 μm의 필터를 통해 미디어를 전달 (모든 오염 피닉스 세포를 제거하기 위해) 및 각질 형성 세포에 2 ml의 배치 (전날 / 잘 75,000 세포에서 도금 : 단계 2.3 참조). 감염 과정을 중재하기 위해 언론에 hexadimethrine 브로마이드 (5 μg의 / ㎖)를 추가합니다. 바이러스 역가는 ~ 1 × 10 7 TU / ㎖이다. <ol><li> RT에서 1 시간 동안 200 XG에서 원심 분리기에서 6 웰 플레이트 스핀. 스핀 후, 바이러스를 포함하는 미디어를 제거하고 KCSFM를 추가하기 전에 1X PBS로 한 번 세포를 씻으십시오. </li></ol></li><li> 제 5 일 : 단계를 다시 2.4로 각질 세포의 동일한 배치를 감염. 참고 : 레트로 바이러스 벡터의 선택 마커가있는 경우 약물을 사용하여 각질을 선택하고 다운 스트림 분석 또는의 Organotypic 문화 등의 용도로 확장합니다. </li></ol> 3. 피부의 Organotypic 문화를 설정 <ol><li> 구축일반 실험실에있는 자료 또는 카세트에서의 Organotypic 카세트 사용자 정의 금속 제조 가게를 통해 할 수있다 (그림 2A - F). 3.5 cm 조직 문화 판에서 이러한 카세트를하려면, 3.5 cm 접시 (그림 2A-B)의 뚜껑의 중심에서 1.0 cm X 1.0 cm 사각형을 제거하는 소작을 사용합니다. 무균 상태에서이 작업을 수행합니다. <ol><li> 명확한 매니큐어 (그림 2C)를 사용하여 카세트 (3.5 cm 접시의 뚜껑)의 바닥에 사각형 못을 연결합니다. 매니큐어 5 분 건조하고 광장 못 (그림 2D)에 쉬고되도록 카세트를 뒤집어하도록 허용합니다. 6cm 접시에 카세트를 놓습니다. 참고 : 광장 예술과 공예 상점의 다양한에서 구입하실 수 있습니다 쐐기. </li></ol></li><li> 각질 세포 성장 배지 DMEM 66 %, 22 %, 햄 F12, 100 유닛 / ㎖ 페니실린 / ㎖ 스트렙토 마이신, 10 % FBS, 2.67 μg의 / ㎖ 아데닌 100 μg의 40 N로 구성된 (KGM)를 준비g / ㎖ 하이드로 코르티손, 10 NG / ㎖ 콜레라 독소, 4.94 / ㎖ μg의 인슐린, 5 μg의 / ㎖ 트랜스페린, 1.36 NG / ㎖ Triiodo-L-로닌, 10 NG / ㎖ EGF 및 10 μg의 / ㎖ 시프로플록사신 염산염. 사용할 준비가 될 때까지 4 ° C에서 0.22 μm의 필터와 저장소를 통해 미디어를 필터링합니다. </li><li> 배 펜 / 연쇄상 구균 + PBS 용액에서 진피를 타고 KGM에서 2 회 반복 한 후 사용하기 전에 이틀 동안 37 ° C에서 KGM에 품어. </li><li> 1.5 cm X 1.5 cm 크기의 조각으로 메스를 사용하여 진피를 잘라 다음의 Organotypic 카세트에 배치합니다. 이 위를 향하게하여 진피의 상부와 진피를 놓습니다. 진피의 정상은 각질 세포 (그림 1B)와 접촉하는면 될 것입니다. </li><li> (그림 3A)를 직면하고있는 진피의 상부면의 Organotypic 카세트의 사각형 구멍에 사전 컷 진피를 놓습니다. </li><li> 집게를 사용하여 위에 진피를 함유하는 전체의 Organotypic 카세트 (도 3b 플립). 얼음 외 기질 용액을 해동. 카세트 당 세포 외 기질 용액 100 μL를 그려 바늘과 주사기를 사용합니다. 진피 (광택면)의 바닥에 5 방울을 추가하고 진피 (그림 3B)에 걸쳐 고른 분포를 확인하기 위해 약간 흔들. </li><li> 상업 세포 외 매트릭스 솔루션을위한 5 분은 완전히 (그림 3C)를 공고히 할 수 있습니다. 응고 후, 다시 위에 카세트를 플립 집게를 사용합니다. 6cm 접시에 KGM의 4 ML을 추가합니다. </li><li> 진피 (그림 3D)를 포함하는의 Organotypic 카세트에 0.5-1000000 유 전적으로 변형 된 각질 세포를 놓습니다. <ol><li> 5 분 동안 10cm 플레이트에 0.05 % 트립신 4㎖를 배치하고 완전 DMEM 배지 4 ㎖로 급냉하여 각화 세포를 Trypsinize. </li><li> 혈구를 사용하여 각질 세포를 계산 한 다음 5 분 동안 200 XG에 스핀 다운. 재현 탁 0.5-1000000 세포는 90 μ로 (사용 가능한 셀의 수)에 따라그리고 KGM의 L은 진피에있는 모든 세포를 놓습니다. 주 : 재생 된 표피의 두께에 차이가 보이는 세포 수를 시작하는 차이에 의해되지 않도록 동일한 실험 진피 내에서 동일한 수의 셀을 배치하는 것이 중요하다. 진피에 미만 50 만 셀을 배치하면 가난한 계층화와 차별화 될 수 있습니다. </li></ol></li><li> 매일의 Organotypic 문화에 KGM 미디어를 변경합니다. 수확 조직 진피에 각질 세포의 배치 후 1-14일. 표피의 전체 계층화 및 분화는 일반적으로 5 일 후에 발생합니다. </li></ol>

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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=Diverse+epigenetic+strategies+interact+to+control+epidermal+differentiation.">Diverse epigenetic strategies interact to control epidermal differentiation.</a> Nat Cell Biol. 14, 753-763 (2012).</li><li>Boxer, L. D., Barajas, B., Tao, S., Zhang, J., Khavari, 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=ZNF750+interacts+with+KLF4+and+RCOR1,+KDM1A,+and+CTBP1/2+chromatin+regulators+to+repress+epidermal+progenitor+genes+and+induce+differentiation+genes.">ZNF750 interacts with KLF4 and RCOR1, KDM1A, and CTBP1/2 chromatin regulators to repress epidermal progenitor genes and induce differentiation genes.</a> Genes Dev. 28, 2013-2026 (2014).</li><li>Jameson, K. 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=IQGAP1+scaffold-kinase+interaction+blockade+selectively+targets+RAS-MAP+kinase-driven+tumors.">IQGAP1 scaffold-kinase interaction blockade selectively targets RAS-MAP kinase-driven tumors.</a> Nat Med. 19, 626-630 (2013).</li><li>Mistry, D. S., Chen, Y., Wang, Y., Sen, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+SNAI2+regulated+genes+in+primary+human+keratinocytes.">Transcriptional profiling of SNAI2 regulated genes in primary human keratinocytes.</a> Genomics data. 4, 43-46 (2015).</li><li>Mali, 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=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339, 823-826 (2013).</li><li>Doebis, C., 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=Efficient+in+vitro+transduction+of+epithelial+cells+and+keratinocytes+with+improved+adenoviral+gene+transfer+for+the+application+in+skin+tissue+engineering.">Efficient in vitro transduction of epithelial cells and keratinocytes with improved adenoviral gene transfer for the application in skin tissue engineering.</a> Transpl immunol. 9, 323-329 (2002).</li><li>Melo, S. 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=Somatic+correction+of+junctional+epidermolysis+bullosa+by+a+highly+recombinogenic+AAV+variant.">Somatic correction of junctional epidermolysis bullosa by a highly recombinogenic AAV variant.</a> Mol Ther. 22, 725-733 (2014).</li><li>Nanba, D., Matsushita, N., Toki, F., Higashiyama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+expansion+of+human+keratinocyte+stem/progenitor+cells+carrying+a+transgene+with+lentiviral+vector.">Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector.</a> Stem cell res ther. 4, 127 (2013).</li><li>Sen, G. L., Reuter, J. A., Webster, D. E., Zhu, L., Khavari, 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=DNMT1+maintains+progenitor+function+in+self-renewing+somatic+tissue.">DNMT1 maintains progenitor function in self-renewing somatic tissue.</a> Nature. 463, 563-567 (2010).</li><li>Shamir, E. R., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+organotypic+culture:+experimental+models+of+mammalian+biology+and+disease.">Three-dimensional organotypic culture: experimental models of mammalian biology and disease.</a> Nat Rev Mol Cell Biol. 15, 647-664 (2014).</li><li>Krejci, N. C., Cuono, C. B., Langdon, R. C., McGuire, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+reconstitution+of+skin:+fibroblasts+facilitate+keratinocyte+growth+and+differentiation+on+acellular+reticular+dermis.">In vitro reconstitution of skin: fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis.</a> J Invest Dermatol. 97, 843-848 (1991).</li><li>Mathes, S. H., Ruffner, H., Graf-Hausner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+skin+models+in+drug+development.">The use of skin models in drug development.</a> Adv Drug Deliv Rev. 69-70, 81-102 (2014).</li></ol>

We have nothing to disclose.

의 Organotypic 문화가 많은 장점을 제공하는 사람의 피부에서 유전자 조작은 일반적으로 2D를 배양 된 세포뿐만 아니라 마우스 모델을 공부합니다. 2D 배양 본래 조직 및 기관에서 발견 삼차원 세포 - 세포 및 세포 - 세포 외 기질의 상호 작용이 부족하다. 최근의 연구는 16 차 종양이 사람의 피부에 많은 유전자 발현 유사성을 보여주는 3 차원 배양 된 세포와 함께 배양 된 2D 및 3D 피부암 세포 ?...

인간 표피뿐만 아니라이 아니라를 보호하는 제 1의 방어선 수분의 손실을 방지하는 불 투과성 장벽 역할 기저막 zone.The 표피로 알려진 세포 외 기질을 통해 하부 진피에 연결 성층 상피이고 외국과 독성 물질 1 몸. 표피의 가장 깊은 층 인 기저층은, 표피 (2)의 나머지를 형성하는 분화 된 자손을 야기 표피 줄기 및 전구 세포를 포함한다. 상피 전구 세포는 분화 된 바와 같이 그들은 가시 층 (3)으로 알려진 분화 된 세포의 제 1 층을 형성하기 위해 위쪽으로 이동한다. 가시 층에서, 세포는 표피의 분화 층 물리적 응력을 견딜 수 있도록 강도를 제공 케라틴 1 내지 10의 발현에 차례. 가시 층 세포는 상기 차별화 된 바와 같이, 그들에 대한 표피의 위쪽으로 이동세포막 아래 조립 keratohyalin와 라멜라 과립의 형성뿐만 아니라, 구조 단백질을 특징으로 과립 층 미터. 세포 분화 과정에서 진행하는 라멜라 과립이 풍부한 층은 각질층 4라는 지질을 형성하는 셀로부터 압출되는 동안, 세포막 밑 단백질은 서로 결합 가교이다. 상피 세포 성장과 분화에 미치는 영향 ~ 인구 5의 20 %의 변화를 포함하는 질병. 따라서,이 과정의 기전을 이해하는 것이 매우 중요하다. 이 질병의 많은 표현이 세포 - 세포 또는 세포 - 매트릭스 접촉에 달려 있기 때문에, 인간의 표피가 3D 환경에서 재구성의 Organotypic 문화 6-10 만들어졌습니다. 이들 방법은 일반적으로 약화 된 인간과 같은 세포 외 기질에 파종 차 또는 변환 된 각화 세포의 사용을 포함진피, 마트 리겔, 또는 콜라겐. 표피 성장과 분화에 중요한 유전자 조절 기전을 이해하기 위하여 케 라티노 유전자 차원 배양 유전자 녹다운 또는 과발현하는 레트로 바이러스 벡터를 통해 조작 된 후 3D로 재구성 될 수있다. 이러한 방법은 종양 11-21 표피 줄기 및 전구 세포자가 재생과 분화뿐만 아니라 진행에 관여하는 유전자를 특성화하는 데 광범위하게 사용되어왔다. 여기서, 레트로 바이러스를 사용하여 배양 물에서, 표피의 Organotypic 유전자 발현을 변경하는 방법에 대한 심도있는 프로토콜이 제공된다.

<table>
	<tbody>
		<tr>
			<td>Human skin</td>
			<td>New York Firefighters Skin Bank</td>
			<td><a href="http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html">http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html</a></td>
		</tr>
		<tr>
			<td>PEN/STREP</td>
			<td>GIBCO</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>amphotropic phoenix cell lines</td>
			<td>ATCC</td>
			<td>CRL-3213</td>
		</tr>
		<tr>
			<td>FUGENE 6 transfection reagent</td>
			<td>Promega</td>
			<td>E2691</td>
		</tr>
		<tr>
			<td>Keratinocyte Media (KCSFM)</td>
			<td>Life Technologies</td>
			<td>17005042</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11995</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>Cambrex</td>
			<td>12-615F</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>10437-028</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma</td>
			<td>A-9795</td>
		</tr>
		<tr>
			<td>Cholera Toxin</td>
			<td>Sigma</td>
			<td>&#160;C-8052</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Calbiochem</td>
			<td>3896</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I-1882</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Invitrogen</td>
			<td>13247-051</td>
		</tr>
		<tr>
			<td>Transferrin&#160;</td>
			<td>Sigma</td>
			<td>T-0665</td>
		</tr>
		<tr>
			<td>Ciprofloxacin Hydrochloride</td>
			<td>Serologicals</td>
			<td>89-001-1</td>
		</tr>
		<tr>
			<td>cautery</td>
			<td>Bovie Medical Corporation</td>
			<td>AA01</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
		</tr>
		<tr>
			<td>Keratin 1 antibody</td>
			<td>Biolegend</td>
			<td>PRB-149P</td>
		</tr>
		<tr>
			<td>square pegs</td>
			<td>Arts and crafts stores</td>
			<td></td>
		</tr>
		<tr>
			<td>human neonatal keratinocytes</td>
			<td>ATCC</td>
			<td>PCS-200-010</td>
		</tr>
		<tr>
			<td>human neonatal keratinocytes</td>
			<td>Cell Applications</td>
			<td>102K-05n</td>
		</tr>
		<tr>
			<td>MSCV retroviral vector</td>
			<td>Clontech</td>
			<td>634401</td>
		</tr>
		<tr>
			<td>LZRS retroviral vector</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pSuper.Retro.Puro Retroviral vector</td>
			<td>Oligoengine</td>
			<td>VEC-PRT-0002&#160;</td>
		</tr>
		<tr>
			<td>hexadimethrine bromide&#160;</td>
			<td>Sigma</td>
			<td>H9268-5G</td>
		</tr>
	</tbody>
</table>

generation of genetically modified organotypic skin cultures using devitalized human dermis

의 Organotypic 문화 기능과 생체 조직의 대응의 생리를 모방 세포 - 세포 연락처 및 세포 - 기질 상호 작용에 중요한 3D 환경의 재구성 할 수 있습니다. 이 충실히 표피 분화와 계층화 프로그램을 되풀이되었습니다의 Organotypic 피부 배양을들 수있다. 기본 인간의 표피 각질 세포는 유전자가 쉽게 과발현 또는 무너 뜨렸다 수 있습니다 레트로 바이러스를 통해 유전자 조작 될 수 있습니다. 이러한 유 전적으로 변형 된 각질 세포는 유전자 전달 경로에 충격을 표피 성장, 분화, 및 질병 진행을 연구하는 모델을 제공하는 강력한의 Organotypic 피부 배양 인간 표피를 재생성하기 위해 사용될 수있다. 여기에 제시된 프로토콜은 약화 된 인간의 진피를 준비뿐만 아니라 유 전적으로 피부의 Organotypic 문화를 생성하기 위해 기본 인간의 각질 세포를 조작하는 방법을 설명합니다. 인간의 피부는 재생 downstr 사용될 수있다이러한 유전자 발현 프로파일, 면역 및 높은 처리량 시퀀싱 하였다 염색질 면역 침전 등 EAM 애플리케이션. 따라서, 이러한 유전자 변형의 Organotypic 피부 문화의 생성은 피부의 항상성 유지에 중요한 유전자의 결정을하실 수 있습니다.

인간 피부의 Organotypic를 생성하는 첫 번째 단계는 표피 진피를 제거하는 것이다. 37 ° C에서 4X 펜 / 연쇄상 / PBS에서 피부의 2 주 인큐베이션 표피 (도 1a)에서 진피의 분리를 허용한다. 표피와 진피를 분리하는 것이 어려운 경우 다른 주 동안 4 배 펜 / 연쇄상 구균에 37 ° C / PBS에서 조직을 배치하고 다시 벗겨 집게를 사용해보십시오. 약화 된 진피에 인간의 표피를 ?...

Watch this Scientific Journal Video about Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis at JoVE.com

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

약화 된 인간의 진피를 사용하여 유전자 변형의 Organotypic 피부 문화의 생성

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

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Developmental Biology

11.2K 조회수.  UCSD Stem Cell Program, University of California, San Diego. The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis. 

비디오: Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

출생 후 신경 발생의 연구 Organotypic 슬라이스 문화

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

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발생학

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

천장 문화 성숙한 지방 세포의 탈분화를 통해 인간 된 지방 줄기 세포의 생성

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

형질, 선택 및 인간 유도 만능 줄기 세포의 콜로니 따기는 AAVS1 세이프 하버 (Safe Harbor)에 GFP 유전자 TALEN는 타겟팅

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

수질 흉선 상피 세포의 증식, 분화 및 무차별 유전자 발현을 지원하는 3D의 Organotypic 공동 문화 모델

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

헤어 유래 각질 세포를 사용하여 통합없는 인간 유도 만능 줄기 세포의 생성

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

에피 솜 벡터를 이용하여 인간 말초 혈액 단핵 세포로부터의 통합없이 유도 다 능성 줄기 세포의 생성

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

차 섬모를 연구에 차 Neurosphere 문화를 사용하여

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the biological output produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis in humans. While studies have explored how modifications to the TSST influence stress responsivity, few studies have created a modified TSST appropriate for vulnerable youth that elicits a significant cortisol stress response. Thus, the current study sought to modify or adjust the TSST in a culturally sensitive way for a vulnerable sample of 14 year-old adolescents. The present study took place within the context of a longitudinal birth cohort study of Mexican American families in California called the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS). The CHAMACOS sample was optimal to test the effectiveness of a modified culturally appropriate TSST, as it is comprised of Mexican American youth, who are often excluded from research. These youths also have experienced significant early life adversity. Example modifications included timed prompts, alternative math tasks, use of same-ethnicity peers as confederates, debriefing immediately after the conclusion of the TSST, and using an unknown youth examiner to administer the debrief. Saliva samples were collected at baseline (after a resting phase), and then again at 15, 30, and 45 min post-TSST onset to assess cortisol concentration. A pilot study of 50 participants (50% female) have been analyzed for cortisol reaction to the TSST. Results confirmed that this modified version of the TSST was successful at eliciting a significant cortisol reaction, with a wide range of variability likely due to individual differences. Goals for modifications and ethnicity considerations are discussed. This study provides the foundation for future research to utilize a modified TSST with vulnerable youth.

Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth.

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

난 모세포의 미세 주입을 통한 유전자 조작 마우스의 생성

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

생성 및 기본 인간 Keratinocytes 성인 피부에서의 경작

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...