우리는 폐를 제공하기 위해 마이애미 대학의 생명 얼라이언스 장기 복구 기관 감사합니다. 우리는 또한 우리는 또한 가브리엘 Gaidosh 감사도 2b 및 3-D, C.에도 3a에 실험에 사용되는 mCherry 바이러스 박사 벤 Gerovac 리사 노박에 나타난 실험에 사용되는 DNA의 준비를 위해 박사 리사 Künzi 감사합니다 이미징 시설에서, 마이애미 대학 안과학 교실. 본 연구는 NIH의 CF 재단과 박사 마티아스 Salathe에 승무원 의학 연구소에서 보조금을 후원하고있다.

1. 1 단계 : HEK 293T의 문화 <ol><li> 고 글루코스 둘 베코 변형 이글 배지 (DMEM)에 10 % (v / v)의 소 태아 혈청 (FBS), 1 % (v / v)의 페니실린 / 스트렙토 마이신을 첨가하여 (HEK 매체로 칭함) HEK 세포 매체를 준비한다. </li><li> 코트 콜라겐 콜라겐 I. 믹스 30 μL I 증류수 2 ㎖에 한 10cm 조직 배양 접시. 접시에 믹스를 확산하고 37 ° C에서 2 시간 5 % CO 2 품어. </li><li> 믹스를 제거하고 15 분 동안 층류 캐비닛의 접시를 건조하게 할 수 있습니다. </li><li> HEK 10 ㎖ 배지에서 1 × 106 세포의 밀도로 피복 된 접시 상에 플레이트 HEK 세포를 37 ℃에서 배양한다, 5 % CO 2. </li><li> HEK 세포 (2~3일 도금 후 그림 1a) 50-60% 합류 경우, 포화 상태의 2 비주얼 평가를 충분한되는 무대로 진행합니다. 어떤 정량화가 필요하지 않습니다. 참고 : HEK 세포 (위 참조) HEK 미디어에서 배양한다. 그들이 포화 상태에 도달 t헤이 분할 (예 : 1/5) 또는 나중에 사용하기 위해 아래로 고정 할 수 있습니다. </li></ol> 2. 2 단계 : HEK 세포의 생산 및 렌티 (정맥 주사)의 농도 참고 : 프로토콜은 BSL-2 미국에서 + (강화 BSL-2) 조건에서 기관 및 정부 규제에 맞춰 실행해야합니다. <ol><li> 칼슘 침전 절차를 사용하여 형질 (리포 펙 타민과 다른 형질 에이전트는 칼슘 침전 절차와 효율적하지 않았다). 다음과 같은 절차가 변경 되더라도 상업 형질 전환 키트의 사용은, 재현성을 보장하는 것이 좋습니다. 실온에 모든 시약을 가져와. 이 날 0입니다. </li><li> HEK293 세포의 각 10cm 접시를 들어, 봉투 DNA pMD2 - VSVG의 4.5 μg의 7.5 μg의 DNA pMDLg / pRRE을 포장, 3.75 μg의 DNA pRSV 반전, 렌티 바이러스 발현 벡터의 10 μg의 DNA, 2 M 칼슘 용액 86 μl를 혼합 및 700 ㎕의 최종 부피로 15 mL의 원심 분리 튜브 내의 멸균(후자의 두 솔루션은 상업 형질 전환 키트에 제공된다). </li><li> 현명한 드롭 (트랜 키트에 HBS) 배 HEPES 완충 식염수 700 ㎕를 (층류 캐비닛) 텍싱하면서 추가하고 30 분 (기다리는 동안 4 단계를 수행)을 실온에서 품어. </li><li> 초기 배양 시간 동안, 슬라이드에 믹스의 예금 5 μL. 천천히 위아래로 초점을 10 배 목표와 현미경으로 액체 방울을 확인합니다. 미세한 침전물은 인산 칼슘 침전 방법을 확인하는 표시한다 (도 1B). </li><li> 혼합하는 상하 HEK 세포 미디어와 피펫 10 ㎖를 첨가하여 30 분 후 침전를 중지합니다. </li><li> 단계 2.1-2.5에서 HEK 세포의 접시를 타고와 매체 (0 일)을 제거합니다. 접시의 가장자리를 따라 조심스럽게 천천히 단계 2.5에서 침전물 솔루션을 추가합니다. </li><li> 다음날 (1 일)에서 분리하고, 침전물을 함유하는 배지를 버리고 승 대체HEK 배지 10 ㎖ 번째. 현미경 미세 침전물 확인 : 그것은 세포 (도 1C 및 d) 사이의 빈 공간에 접시에 볼 수 있어야합니다. </li><li> 제 2 일에, 40 %의 폴리에틸렌 글리콜 3.8 ㎖ (PEG) 용액을 함유하는 15 mL의 원심 분리 튜브에 0.45 ㎛의 주사기 필터를 통해 주사기, 필터 매체를 수집한다. 합니다 (PEG가 형성 침전,하지만 더 이상 오일보다 용) 바이러스 컬렉션의 모든 때까지 완료 반전 5 배는 최소 24 시간 동안 4 ℃에서 혼합하지 않고 저장합니다. </li><li> 일 3 일 4 4. 단계를 반복 2.8 HEK 매체를 대체하지만 10 %의 표백제로 오염을 제거하고 요리를 폐기하지 않습니다. </li><li> 4 ° C에서 20 분 동안 1,650 XG에 원심 분리기 모든 컬렉션 튜브 (함께 5 일에 일반적으로 모든 컬렉션) 바이러스 펠렛합니다. 제거하고 뜨는을 폐기하고, 수집 및 PEG 상등액의 나머지를 제거하기 위해 4 ° C에서 5 분 동안 1,650 XG에 다시 스핀. </li><li> ResusPEND 암포 테리 신 B (8)가없는 기관지 상피 성장 배지 (BEGM) 200 ㎕를 각 튜브의 펠렛. 함께 수영장과 200 μL에서 나누어지는. -80 ° C에서 보관 바이러스. 에이즈-1 개그 (P24) 항원 ELISA 분석을 수행하기 위해 추가로 10 μl를 나누어지는. </li><li> 시판 ELISA 키트를 사용하여 제조사의 프로토콜에 따라 P24 항원 분석을 수행한다. 분석은 pg / ml 인에서 바이러스 P24의 추정치를 제공합니다. </li></ol> 3. 3 단계 : 문화 차 HBE 세포 <ol><li> 10 ㎖ BEGM 매체에 플레이트 HBE 세포 I은 10cm 조직 배양 접시 코팅 콜라겐 1 × 106 세포의 밀도로 (100 μL 암포 테리 신 B와 통로 0 세포 수 진균의 오염을 제거하기 위해 사용되는 경우). 37 ° C에서 품어 5 % CO 2. </li><li> 다음날, 배지를 제거하고 사일 총 (100 μL 통로 암포 테리 신 B와 0 세포)를 10 ㎖ BEGM 교체. 인큐베이터로 돌아갑니다. </li><li> 4 D 후,AYS는 매일 암포 테리 신 B를 변경 매체없이 만 BEGM를 사용합니다. </li><li> 세포가 80-90% 합류 (그림 2a; ~ 칠일 도금 후) 때, 전달 진행 (전달하기 전에 준비를 위해 3.5에주의). 또, 포화 상태의 시각적 평가는 충분하다. 어떤 정량화가 필요하지 않습니다. </li><li> 전달에 앞서, 외투 콜라겐 IV 용액 투과성지지 인서트는 멸균 증류수로 1/10 희석 하였다. 각 삽입에 200 μl를 추가하고 층류 캐비닛 건조 하룻밤을 할 수 있습니다. </li></ol> 4. 4 단계 : HBE 세포의 형질 도입 <ol><li> 용지를 꺼내 인산염 완충 생리 식염수 (EDTA / PBS)에 1 mM의 에틸렌 디아민 테트라 아세트산 0.1 % 트립신 3 ㎖를 추가하고 37 ° C에서 5 분 5 % CO 2를 품어. 모든 세포가 분리되어 있는지 확인하기 위해 현미경 (10X 목적)에서 확인하십시오. 그렇지 않은 경우, 추가로 5 분 동안 인큐베이터로 돌아갑니다. </li><li> 모든 세포가 분리되는 경우 (3)을 추가대두 트립신 억제제 ㎖ (SBTI 1 ㎎ / ㎖)을 혼합하고 15 mL의 원심 분리 튜브에 이송. 실온에서 5 분 동안 460 XG에서 회전하여 세포 펠렛. </li><li> BEGM 3 ㎖에 뜨는에 resuspend 펠렛을 제거하고 계수를 진행합니다. </li><li> BEGM 400 μL (표 2 참조)에서 하나 12mm 투과성지지 인서트 당 2 × 105 세포의 비율로 재현 탁 HBE 세포 (이제 통로 1 P1). 형질한다 투과성지지 인서트의 양에 기초하여 이들 숫자를 추정. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 표면 </td><td> 표면적 </td><td> 세포 수 </td><td> 미디어 </td><td> 볼륨 (미디어) </td></tr><tr><td> 10cm 접시 </td><td> 52.7 cm (2) </td><td> 1 × 10 (6) </td><td> BEGM </td><td> 10 ml의 </td> &lt;/ TR&gt; <tr><td> 12mm 필터 </td><td> 1.13 cm (2) </td><td> 2 × 10 5 </td><td> BEGM </td><td> 400 μL </td></tr><tr><td> 24mm 필터 </td><td> 4.52 cm (2) </td><td> 8 × 105 </td><td> BEGM </td><td> 800 μL </td></tr></tbody></table> 표 2 : 세포 수에 따라 미디어 외삽. <ol start="5"><li> (4)의 감염 (MOI) 계수의 다양성을 이용하여 세포 현탁액으로 바이러스가 적절한 볼륨. </li><li> 2 μg의 / ml의 최종 농도 hexadimethrine 브로마이드를 추가합니다. </li><li> 꼭대기 실에 투과 지원 인서트 당 믹스 (BEGM, HBE 세포, 바이러스 및 hexadimethrine 브로마이드) 400 μl를 분배하고, 기저 구획에 혼자 BEGM 1 ㎖를 추가합니다. 제어 및 테스트 퓨로 마이신 선택 (해당하는 경우)와 같은 바이러스없이 37 ° C, 5 % CO 2. 항상 판 HBE 세포에서 밤새 품어. </li><li> 다음 날, REM 수면에비켜 혀끝과 기저 미디어, PBS로 세척하고, 공기 - 액체 인터페이스 (ALI) 미디어 8 두 구획을 교체합니다. </li><li> 세포가 컨 플루 언트 될 때, (공기 - 액체 계면을 확립라고도 함) 꼭대기 유체를 제거한다. 그들은 완전한 성숙에 도달 할 때까지 바닥 구획 매일의 매체 (ALI)를 변경 계속; 보통 3 ~ 4 주 후. 주 : 렌티 바이러스 벡터는 퓨로 마이신과 같은 선별 유전자를 포함하는 경우, 세포를 선택 농도 곡선에있어서, 퓨로 마이신을 첨가하여 선택 될 수있다. HBE 세포, 1 μg의 / ml의 농도가 형질 도입 된 세포의 선택 일치 이상적인 것으로 결정되었다. 그러나,이 농도는 다른 세포 또는 조건과 다를 수 있습니다 곡선을 살해에 의해 결정되어야한다. 미디어가 선택 유전자의 전체 표현을 할 수 있도록 나중에 ALI 미디어 또는 (2-3 일)로 대체 한 후 퓨로 마이신의 추가는 이른로 하루를 시작하세요. 하나의 삽입 그 시간에 퓨로 마이신을 추가해야합니다같은 형질 도입되지 않았다. 세포가 완전히 분화까지 퓨로 마이신이 첨가 될 수있다. </li></ol>

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J., Ivonnet, P., Dennis, J. S., Conner, G. E., Salathe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta1+and+cigarette+smoke+inhibit+the+ability+of+beta2-agonists+to+enhance+epithelial+permeability.">Transforming growth factor-beta1 and cigarette smoke inhibit the ability of beta2-agonists to enhance epithelial permeability.</a> Am J Respir Cell Mol Biol. 52, 65-74 (2015).</li><li>Geraerts, M., Willems, S., Baekelandt, V., Debyser, Z., Gijsbers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+lentiviral+vector+titration+methods.">Comparison of lentiviral vector titration methods.</a> BMC Biotechnol. 6, (2006).</li><li>Schneiderman, S., Farber, J. L., Baserga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+decreasing+the+toxicity+of+polyethylene+glycol+in+mammalian+cell+hybridization.">A simple method for decreasing the toxicity of polyethylene glycol in mammalian cell hybridization.</a> Somatic Cell Genet. 5, 263-269 (1979).</li><li>Schmid, 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=Soluble+adenylyl+cyclase+is+localized+to+cilia+and+contributes+to+ciliary+beat+frequency+regulation+via+production+of+cAMP.">Soluble adenylyl cyclase is localized to cilia and contributes to ciliary beat frequency regulation via production of cAMP.</a> J Gen Physiol. 130, 99-109 (2007).</li><li>Manzanares, D., 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=Airway+Surface+Dehydration+by+Transforming+Growth+Factor+beta+(TGF-beta)+in+Cystic+Fibrosis+Is+Due+to+Decreased+Function+of+a+Voltage-dependent+Potassium+Channel+and+Can+Be+Rescued+by+the+Drug+Pirfenidone.">Airway Surface Dehydration by Transforming Growth Factor beta (TGF-beta) in Cystic Fibrosis Is Due to Decreased Function of a Voltage-dependent Potassium Channel and Can Be Rescued by the Drug Pirfenidone.</a> J Biol Chem. 290, 25710-25716 (2015).</li><li>Manzanares, D., 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=IFN-gamma-mediated+reduction+of+large-conductance,+Ca2+-activated,+voltage-dependent+K++(BK)+channel+activity+in+airway+epithelial+cells+leads+to+mucociliary+dysfunction.">IFN-gamma-mediated reduction of large-conductance, Ca2+-activated, voltage-dependent K+ (BK) channel activity in airway epithelial cells leads to mucociliary dysfunction.</a> Am J Physiol Lung Cell Mol Physiol. 306, L453-L462 (2014).</li><li>Chen, X., 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+soluble+adenylyl+cyclase+form+targets+to+axonemes+and+rescues+beat+regulation+in+soluble+adenylyl+cyclase+knockout+mice.">A soluble adenylyl cyclase form targets to axonemes and rescues beat regulation in soluble adenylyl cyclase knockout mice.</a> Am J Respir Cell Mol Biol. 51, 750-760 (2014).</li><li>Ransford, G. 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=Pannexin+1+contributes+to+ATP+release+in+airway+epithelia.">Pannexin 1 contributes to ATP release in airway epithelia.</a> Am J Respir Cell Mol Biol. 41, 525-534 (2009).</li></ol>

The authors have nothing to disclose.

여기에 설명 된 프로토콜은 100 % 전달 효율에 가까운 보장합니다. 그러나,이 목표를 달성하기 위해 중요한 과정에서 단계가있다. 예를 들어, 형질 전환 과정은 형질 믹스 석출물의 존재 (드롭) 또는 접시에 형질 도입 (도 1b 및 d) 다음날 좋은 형질 대한 중요한 양이다. 침전 또는 응집의 존재 유무는 형질 전환 시약 중 하나의 누락, pH가 문제 또는 불량 플라스미드 제조에 기...

복제 결함, pseudotyped 렌티 바이러스의 개발 (LV)는 관내 1 유전자를 전달하기 위해 안전하고 효율적인 방법으로 연구자를 제공하고 있습니다. 그들의 장기 표현이 LV는 또한 생체 내에서 2,3- 치료제의 전달을 위해 사용될 수있다. LV의 생산은 여러 플라스미드 인간 배아 신장 (HEK) 세포의 공동 - 형질 필요 전이 벡터 포장 개그 / 폴, 포장의 회전을하고, 수 포성 구내염 바이러스 당 단백질 (VSV-G) 플라스미드 봉투. 레브 유전자하여 복제 가능 렌티 바이러스를 생산하는 재조합의 가능성을 감소시키는, 별도의 패키징 플라스미드 인코딩되기 때문에 제 3 세대 포장 시스템은 제 2 세대 시스템보다 안전한 것으로 간주된다. 2 세대 포장 시스템 다섯 배 더 높은 총 전달 유닛을 수득하지만, 원래의 바이러스 게놈의 분할 만드는3 세대 시스템은 최소한 3,4- 전달 레벨이 감소하고있다. 세포주를보다 쉽고 효율적으로 형질 도입 될 수 있지만 이러한 생체 더 대표는 5,6 어떤 조직이 있기 때문에, 연구자들은 일차 전지에 그들의 초점을 이동하고있다. 그러나, 일차 전지는 전달하는 내화물이다. 완전히 분화 ​​된기도 상피 세포의 형질 도입이보고되었지만, 전체 효율은 15 % 7을 초과하지 않았다. 높은 역가의 정맥 주사의 생산을 가능하게하는 프로토콜은 여기에 설명한다. 단계별 접근법은 기본 HBE 세포로 거의 100 % 형질 도입 효율을 달성하기 위해 설명된다. 구체적으로는, 프로토콜은 3 성공 쓸모 최적 배양 조건을 설명한다. 간단히, HEK의 293T 세포는 향상된 녹색 형광의 EF-1 프로모터 구동 식으로 렌티 바이러스 발현 벡터 pCDH를 사용하여 형질단백질 (eGFP는) mCherry 또는 적색 형광 단백질, 및 제 3 세대의 패키징 시스템. 미디어에 발표 정맥 주사 이후 24 시간 (72)에 수집된다. 바이러스 입자는 P24 항원의 효소 면역 분석법 (ELISA) 키트를 사용하여 역가를 추정하기 전에, 폴리에틸렌 글리콜 (PEG), 바이러스의 농도를 더욱 간편한 방법에 집중된다. 4 (MOI) 인자 감염 다중도에서 (트립신 처리 후)에 부착하는 동안 계속해서, 미분화 차 HBE 셀 (hexadimethrine 브로마이드를 첨가하고, 16 시간 동안 배양하고, 증식 배지 (기관지 상피 성장 배지 또는 BEGM) (8)에 형질 도입되고 밤새). 세포는 분화 할 수있다. 바이러스 유전자는 (설명을 참조 적절한 프로모터를 사용하는) 장기간 발현되므로, 발현은 pseudostratified기도 상피 세포로의 분화에 걸쳐 유지된다 또는 분화 후 (유도 성 프로모터를 사용하여) 유도 될 수있다.

<table><tbody><tr><td>HEK293T/17 cells</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Sigma</td><td>12306C</td><td>use at 10%</td></tr><tr><td>DMEM</td><td>Thermo Scientific</td><td>11995-040</td><td></td></tr><tr><td>Penicillin/Streptomycin (100x)</td><td>Thermo Scientific</td><td>15140-122</td><td>use at 1x</td></tr><tr><td>Collagen I</td><td>BD Biosciences</td><td>354231</td><td>dilute 1:75 in distilled water</td></tr><tr><td>Calphos</td><td>Clontech</td><td>631312</td><td>Transfection kit containing 2 M Calcium solution, 2x HEPES-Buffered solution (HBS) and sterile H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Polythylene glycol 8000</td><td>VWR scientific</td><td>101108-210</td><td>stock of 40%</td></tr><tr><td>Amphotericin B</td><td>Sigma</td><td>A9528</td><td>use at 1x</td></tr><tr><td>Trypsin</td><td>Sigma</td><td>T4799</td><td></td></tr><tr><td>Soybean trypsin inhibitor</td><td>Sigma</td><td>T9128</td><td></td></tr><tr><td>Transwell 12 mm</td><td>Corning</td><td>3460</td><td></td></tr><tr><td>Collagen IV</td><td>Sigma</td><td>C7521</td><td>dilute 1:10 in distilled water</td></tr><tr><td>Hexadimethrine bromide</td><td>Sigma</td><td>H9268</td><td>stock of 2 mg/ml</td></tr><tr><td>Puromycin (10.000x)</td><td>Thermo Scientific</td><td>A11138-03</td><td>use at 1x</td></tr><tr><td>Alliance HIV-1 p24 ELISA</td><td>PerkinElmer</td><td>NEK050001KT</td><td></td></tr><tr><td>F12</td><td>Thermo Scientific</td><td>11765-054</td><td></td></tr><tr><td>pCDH-EF1-MCS-IRES-Puro</td><td>System Biosciences</td><td>CD532A-2</td><td>lentiviral expression vector</td></tr><tr><td>pMD2-VSVG</td><td>Addgene</td><td>12259</td><td>packaging DNA</td></tr><tr><td>pMDLg/pRRE</td><td>Addgene</td><td>12251</td><td>packaging DNA</td></tr><tr><td>pRSV-rev</td><td>Addgene</td><td>12253</td><td>packaging DNA</td></tr></tbody></table>

optimal lentivirus production and cell culture conditions necessary to successfully transduce primary human bronchial epithelial cells

호흡기 세포 분화 및 기능의 처리를 연구하는 유용한 모델을 공기 - 액체 계면 상태를 제공하여 기본 인간 기관지 상피 (HBE) 세포의 시험 관내 배양한다. 지난 몇 년 동안, 유전자 전달에 대한 렌티 바이러스 벡터의 사용은 일반적인 관행이되었다. 특정 비 HIV 의사 입력 렌티 완전히 분화 된기도 상피 세포의 형질 도입의보고가 있지만, 전체의 전달 효율이 일반적으로 15 % 미만이다. 여기에 제시된 프로토콜은 신뢰할 수 있고 효율적인 방법 렌티 바이러스를 제조하고,이 차 인간 기관지 상피 세포를 형질 도입하기를 제공한다. 세포가 부착하는 동안 4 감염 인자의 다양성과 함께, 미분화 기관지 상피 세포, 기관지 상피 성장 배지에서 형질을 사용하여 100 %에 가까운 효율을 제공한다. 이 프로토콜은, 단계적으로 높은 역가 렌티 바이러스 벡터 및 제의 제조와 농도를 설명전자 전달 방법. 이는 일차 인간 기관지 상피 세포 transductions 고효율을 달성하기 위해 최적 배양 조건을 결정 실험을 설명한다.

도 1은도 1a. 형질 감염 과정 셀 솔루션을 평가하는 다른 주요 단계를 도시 전에 바이러스 생산을위한 형질 전환에 HEK293T 세포의 최적의 포화 상태를 나타낸다. 이는 세포가 접시에 걸쳐 균등하게 분배하는 것이 중요하다.도 1b는 명 시야 광학계 및 10X 대물 렌즈를 이용하여 형질 전환 혼합물의 드롭 침전물을 보여주?...

Watch this Scientific Journal Video about Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells at JoVE.com

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

최적의 Lentivirus 생산 및 세포 배양 조건에 필요한 성공적으로 형질 도입 기본 인간 기관지 상피 세포

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

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19.2K 조회수.  University of Miami, Miller School of Medicine. Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

비디오: Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

포장 HIV 또는 FIV 기반 Lentivector 식 구조 및 VSV-G Pseudotyped 바이러스성 입자의 형질 도입

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

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Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

영양막 줄기 세포로 쥐 배아 섬유 아세포의 직접적인 변환을위한 프로토콜

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

발생학

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 ...

유전학

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

항생 물질 규제 세포주 특히 수지상 세포를 대상으로 높은 titer의 렌티 바이러스 벡터를 생성

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

확산 및 Murine 골수성 전조 32D/G-CSF-R의 셀

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Explants에서 들어라 기본 인간 기관지 상피 세포

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

생물학

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

향상된 Biosafety에 대한 고분자 - complexed Ecotropic Lentivirus과 인간의 세포의 형질 도입

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

고병원성 H5N1 및 조류 H7N9 바이러스의 엔벨로프 당단백질을 가진 고적퇴력 감염성 인플루엔자 슈도타입 입자 의 생산

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in na&#239;ve human populations.

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

호흡기 바이러스 감염 동안 선천적 면역 세포 활성화의 연구를 위한 비접촉식 공동 배양 모델

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.