이 프로젝트에 대한 자금은 재생 의료에 대한 캘리포니아 연구소에 의해 제공되었다.

1. Lentivirus production, harvest, and freezing
<ol>
	<li>Consult your institutional safety official before beginning this protocol, and follow their recommended safety guidelines.</li>
	<li>Day 1. Start with healthy, rapidly growing 293T cells to produce virus. Plate the cells at 5 x 106 
	cells per 10 cm plate. Use antibiotic-free 293T medium (high-glucose DMEM with 10% FBS and 4 mM L-glutamine) for virus production.</li>
	<li>Day 2. In the late afternoon, transfect 293T cells as follows (numbers given are for one 10 cm plate). 
	Let all reagents warm up to room temperature. Pipette 375 &mu;l OptiMEM into a microcentrifuge tube, then add 25 &mu;l 
	Fugene HD. Do not allow undiluted Fugene to contact the surface of the tube.</li>
	<li>In a microcentrifuge tube, mix:
		<ul>
			<li>5 &mu;g transfer plasmid (Slc7a1, target vector, or fluorescent control vector)</li>
			<li>3.75 &mu;g packaging plasmid (pCMV-dR8.91 or psPax2)</li>
			<li>1.25 &mu;g envelope plasmid (pMD2.G for pantropic, pHCMV-EcoEnv for ecotropic)</li>
			<li>serum-free OptiMEM to 100 &mu;l</li>
		</ul>
	</li>
	<li>Combine the two tubes and incubate the mixture 20-30 minutes at room temperature.</li>
	<li>Change the medium on the 293T cells to 10 ml fresh antibiotic-free 293T medium. Add the Fugene/plasmid mix 
	drop-wise to the plate and incubate overnight at 37&deg;C, 5% CO2.</li>
	<li>Day 3. Change medium on the 293T cells to 10 ml fresh antibiotic-free 293T medium. Be gentle, 
	because 293T cells adhere only loosely and can slough off by media pipetted too forcefully onto the 
	monolayer. Incubate for two days in a humidified incubator for virus production.</li>
	<li>Day 5. Harvest virus and filter with a 0.45 mm low protein binding filter. Use immediately or dispense 
	into single-use aliquots and freeze at -80&deg;C. Frozen pantropic and ecotropic virus should be re-titered after it has 
	been stored for six months and one month, respectively.</li>
	<li>Titer the virus as follows, using cells that are relatively amenable to transduction. Transduce cells with fluorescent control virus overnight using serial dilutions (e.g. 1:10, 1:100, 1:1000) in fresh medium with 6 &mu;g/ml PB. Change to fresh medium the next day. Allow the cells two days after transduction to begin expressing the fluorescent protein, then determine the fraction of transduced cells by FACS. Calculate the titer in transforming units (TU) per ml, based on dilutions that yield &lt; 15% transduction to minimize multiple transduction events. 
	<img src="/files/ftp_upload/2822/2822eq1.jpg" alt="Equation 1" />
	</li>
</ol>
2. Transduction of human target cells with murine retrovirus receptor Slc7a1
<ol>
	<li>Select a multiplicity of infection (MOI) = 2 to ensure that most cells will be transduced. For target cells that are resistant to transduction such as HDFs, a higher MOI will be required; we dilute the viral supernatant only 1:2 to achieve transduction of a majority of the cells.</li>
	<li>Transduce target cells overnight with viral supernatant diluted in fresh culture medium with 6 &mu;g/ml PB, which increases transduction by enhancing electrostatic interaction between the virus and target cell.1 Ideally one should use a minimal volume of virus, such as 1 ml for a 35 mm plate, because diffusion is a limiting factor in transduction efficiency. </li>
	<li>Culture cells for at least 48 hours after removing virus before transducing them with ecotropic virus, in order to ensure sufficient expression of the Slc7a1 receptor.</li>
	<li>(Optional) It is possible to use blasticidin to select for stably transduced cells expressing Slc7a1, if desired. A blasticidin kill curve should be generated in advance to determine the lowest effective concentration, generally between 2 &#x2013; 10 &mu;g/ml, that kills all untransduced target cells in 7 days. About 2 days after removing the Slc7a1 virus, switch the transduced cells to medium containing blasticidin. Culture the cells in blasticidin-containing medium for 7 days, at which point all remaining cells will stably express the retrovirus receptor at which point they can be transduced with ecotropic virus as well as banked for future use as a stable Slc7a1-expressing line.</li>
</ol>
3. Polymer complex titration to determine toxicity
<ol>
	<li>Day 1. Plate cells at 5000 cells/well in a 96-well plate, allowing at least triplicate wells for each experimental group. Include untransduced cells in complete medium to provide a baseline value for healthy cells, as well as samples consisting of cells in serum-free medium to induce growth arrest as a control. In parallel, plate cells in an appropriate format (such as a 24-well plate) for microscopic or FACS-based analysis of transduction efficiency in each experimental condition.</li>
	<li>Day 2. Transduce cells in plates set up for both the MTT and FACS analyses with virus encoding GFP, at an MOI of 0.5 to transduce ~40% of target cells. Test varied concentrations of CS/PB (e.g. 50, 100, 200, 400, 600, and 800 mg/ml of each component), as well as 6 &mu;g/ml PB, to determine optimal amounts. Generate polymer complexes as described in Part 4, below.</li>
	<li>Day 3. Remove virus-containing medium and replace with fresh medium. Return the plates to a humidified incubator.</li>
	<li>Day 5. Analyze FACS plate to determine the transduction efficiency with each polymer concentration. </li>
	<li>Day 6. Remove the medium from all wells of the MTT plate and replace with 100 &mu;l MTT solution (1 mg/ml MTT in complete medium). Culture for three hours in a humidified incubator to allow MTT to be reduced in the mitochondria of metabolically active cells.</li>
	<li>Remove MTT solution and replace with 200 ml MTT solvent (0.1 N HCl, 0.1% Igepal CA-630 in isopropanol). </li>
	<li>Incubate for two hours at room temperature or until all of the purple MTT formazan precipitate is dissolved. Read the absorbance on a microplate reader at 570 nm, subtracting the background reading at 690 nm. </li>
	<li>Select the optimal polymer concentration that produces maximum enhancement of transduction, as determined by FACS, with minimal effect on metabolic activity, as determined by MTT. For HDFs, we selected 100 &mu;g/ml CS/PB based on the data shown in the Representative Results, below.</li>
</ol>
4. Ecotropic transduction with polymer complexation
<ol>
	<li>Prepare sterile-filtered stock solutions of PB and chondroitin sulfate at 20 mg/ml in water. Aliquot and store at -20&deg;C.</li>
	<li>Pipette viral supernatant into a microcentrifuge tube and dilute to the desired final concentration (e.g. MOI of 2) with fresh culture medium. Add equal volumes of PB and chondroitin sulfate (concentrations determined in step 3 above) in succession, flicking the tube to mix after each addition. The viral mixture will immediately become cloudy as precipitates form. Incubate the viral mixture at room temperature for 5 minutes to allow complex formation.</li>
	<li>Remove media from the receptor-expressing target cells, replace with the viral mix, and incubate overnight at 37&deg;C, 5% CO2. After removing the viral mix, wash the surface of the cells twice with PBS to aid in removing viral complexes. Complete removal is not necessary; we have observed no adverse effects on cell health from residual polymer complexes.</li>
</ol>
5. Verifying specificity of ecotropic transduction
<ol>
	<li>When producing ecotropic virus for the first time, it is prudent from a safety perspective to verify that the virus is unable to transduce unmodified human cells. Transduce human cells with ecotropic lentivirus at relatively high concentration (only a 1 to 2-fold dilution of viral supernatant in fresh medium) overnight with 6 &mu;g/ml PB or optimized CS/PB concentration. </li>
	<li>Remove virus-containing medium and replace with fresh medium. After incubating cells for 2 days, validate transgene expression by FACS using fluorescent vectors and/or by RT-PCR with transgene-specific primers.</li>
</ol>
6. Representative results:
Fig. 1A shows the lentivirus production process, and Fig. 1B shows transduction of human cells with ecotropic lentivirus, including pre-transduction with murine retrovirus receptor Slc7a1. For titering virus, we use a human rhabdomyosarcoma cell line stably transduced with Slc7a1 (Slc-hRMS). When using fluorescent control vectors to monitor transduction efficiency, we routinely achieve > 90% transduction efficiency of Slc-hRMS with ecotropic virus, as shown in Fig. 2A and 2B. Transduction rates of HDFs are generally lower because the cells have not been blasticidin-selected for receptor expression (Fig. 2C). Titers of ecotropic lentivirus are generally 10-20% of VSV-pseudotyped virus when measured on Slc-hRMS (Fig. 2D).
One freeze-thaw cycle reduces titer of ecotropic virus by 16 - 3%, equal to titer loss during a single freeze-thaw cycle of VSV-pseudotyped virus (p > 0.05). Contrary to previous reports,2 we observe no positive effect on virus titer from flash freezing in dry ice. Rather, we achieve higher post-thaw titers of either pseudotype by simply placing tubes of virus into a -80&deg;C freezer (data not shown). 
The MTT viability assay allows sensitive detection of growth arrest in target cells. Transduction with virus plus concentrations of CS/PB up to 800 &mu;g/ml has no effect on HDF metabolism (Fig. 3A). Exposure to CS/PB without virus also has no toxic effect on cells (data not shown). FACS analysis of HDFs transduced with virus plus various concentrations of CS/PB shows enhancement of transduction compared to PB alone (Fig. 3B). The maximum enhancement occurs at 100 &mu;g/ml CS/PB (Fig. 3C), several-fold lower than previously reported values.3 Thus, it is important to optimize conditions for any given target cell type.
Complexation with CS/PB enhances the observed titer roughly 3-fold in Slc-hRMS (Fig. 4A, p &lt; 0.01). In practice, this yields a greater effect on transduction efficiency at low virus concentrations than at higher concentrations (Fig. 4B), which is most likely due to multiply transduced cells and receptor saturation at higher virus concentrations.
Transduction with ecotropic virus is specific for cells expressing murine retrovirus receptor. When transducing unmodified human cells with ecotropic virus, we have not observed fluorescence greater than the untransduced background whether using PB or CS/PB, as shown in Fig. 5A. Microscopically, HDFs show no transduction in the absence of receptor (Fig. 5B) while pre-transduction with receptor results in fluorescent cells (Fig. 5C).
<img src="/files/ftp_upload/2822/2822fig1.jpg" alt="Figure 1" /> 
Figure 1. Schematic view of (A) the virus production process and (B) transduction of human cells with murine retrovirus receptor followed by ecotropic lentivirus.
<img src="/files/ftp_upload/2822/2822fig2.jpg" alt="Figure 2" /> 
Figure 2. High-efficiency transduction of human cells with ecotropic lentivirus encoding GFP. Cells were pre-transduced with murine retrovirus receptor Slc7a1. (A) Human rhabdomyosarcoma cell line blasticidin-selected for Slc7a1 (Slc-hRMS), transduced (blue) or untransduced (pink) with GFP. (B) Fluorescent micrograph of ecotropic-transduced Slc-hRMS (4X). (C) HDFs transduced (blue) and untransduced (pink) with ecotropic GFP lentivirus; cells were transduced with Slc7a1 two days prior to ecotropic transduction. (D) Titer of VSV-G pseudotyped and ecotropic lentivirus, measured on Slc-hRMS.
<img src="/files/ftp_upload/2822/2822fig3.jpg" alt="Figure 3" /> 
Figure 3. Optimizing chondroitin sulfate/polybrene (CS/PB) concentrations in human dermal fibroblasts. (A) Viability of HDFs measured by MTT assay after transduction in the presence of PB only or with varied concentrations of CS/PB. (B) Enhancement of transduction efficiency in HDFs by different concentrations of CS/PB, measured by FACS. (C) Quantification of transduction enhancement, showing that maximum transduction occurs at 100 &mu;g/ml CS/PB.
<img src="/files/ftp_upload/2822/2822fig4.jpg" alt="Figure 4" /> 
Figure 4. Effect of 400 &mu;g/ml CS/PB on transduction of Slc-hRMS with ecotropic lentivirus. (A) Titer enhancement, and (B) fold change in transduction rate compared to 6 &mu;g/ml polybrene alone.
<img src="/files/ftp_upload/2822/2822fig5.jpg" alt="Figure 5" /> 
Figure 5. Lack of ecotropic transduction of HDFs in the absence of murine retrovirus receptor Slc7a1. (A) FACS plot of BJ human fibroblasts, untransduced (pink) or transduced (blue) with ecotropic GFP lentivirus in both cases in the absence of receptor Slc7a1. BJ human fibroblasts transduced with ecotropic GFP lentivirus, without (B) and with (C) pre-transduction with receptor Slc7a1 (4X).

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관심 없음 충돌 선언하지 않습니다.

몰로니 murine의 백혈병 바이러스 (MLV)과 그 수용체 mSlc7a1에 따라 재조합 ecotropic gammaretrovirus 잘 연구하고 널리 없습니다 murine 세포 transgenes를 제​​공하기 위해 20여 년 동안 사용되고 보내고있다. Ecotropic gammaretrovirus도 인간의 세포 oncogenes를 제공하기 위해 최근 사용되고 있습니다,. 셀룰러 프로그래밍의 맥락에서, mSlc7a1의 사용은 잘 설립 인간 oncogenes을 품고 amphotropic 바이러스의 생성을 피하기 위해 4,5 단, lentivirus은 gammaretrovirus에 비해 상당한 장점을 제공합니다 lentiviral 사전 통합 복잡한 허용하기 때문에 내화물 세포 집단, 종종 프로그래밍을 위해 바람직 목표물 6를 포함한 기본 세포를 transducing가 아닌 나누어 세포의 형질 도입 7. Lentiviruses는 바이러스 tropism, 독성, 및 기타 특성 8. MLV - pseudotyped ecotropic의 lentivirus은 마우스 세포, 9 transduce하는 데 사용되었지만 거의 인간의 세포에 사용되지 않은 내용을 변경하기 위해 MLV를 포함한 다양한 pseudotypes 수십, 함께 제작되었습니다 10. 우리는 따라서 인간의 세포에 세포 프로그래밍 요소를 포함하여 알려지거나 의심 oncogenes을, 제공하는 안전하고 비용 효율적인, 그리고 고효율 차량으로 MLV - pseudotyped ecotropic lentivirus의 사용을 제안합니다. 오히려,이 프로토콜은 암 관련 바이러스 자체 접종 잠재력에서 연구자를 단열, pantropic 바이러스에서 oncogene (들)을 분리, 그것은이 프로토콜은 완전히 pantropic lentivirus을 생산하고 사용할 필요성을 제거하지 않습니다에 중요합니다. 단백질 mSlc7a1과 인간 homologue의 hSlc7a1는 ubiquitously 알려진 tumorigenicity 또는받는 사람 세포에 선택 성장 우위를 부여하는 능력, amphotropic 바이러스에 설립에 대한 상대적으로 낮은 위험이 11 만들기 mSlc7a1 아니요 아미노산 운반을 표명하고 있습니다. 이것은 추가 단계 필요 biosafety 수준의 전용 바이러 스나 조직 문화 시설 부족 실험실에서 특히 유용할 수 있습니다. 어떤 상황에서는 완전히 대상 세포에 직접 Slc7a1이 플라스미드 transfecting에 의해 pantropic 바이러스의 사용을 제거하는 것이 가능 수 있지만,이 기술의 유용 대상 될 많은 세포는 transfection에 내화물 있습니다. 대안으로 분리할 수있는 능력 blasticidin 선택하여 세포를 Slc7a1는 - transduced 것은 VSV - G pseudotyped lentivirus가 ecotropic 바이러스가 많은 이들 세포를 transduce하기 위해 일상적으로 사용할 수있는 이후 수용체 - 표현 세포의 주식을 생성 한 번 사용될 수있다는 것을 의미 실험. 연구원에 관계없이 항상 그 tropism의 모든 lentivirus 작업을 위해 제도적 안전 지침을 따라야합니다. 이 프로토콜과 함께 실현 ecotropic 바이러스 Titers 이전 연구와 합의에 일반적으로 VSV - pseudotyped 바이러스, pantropic 바이러스 titer의 10~20%보다 적당히 낮은 9. 이러한 titers가 안정 Slc7a1와 transduced 인간의 세포로 측정되었다, 그래서 낮은 ecotropic 바이러스에 대한 관찰 titer는 대상 세포에있는 수용체 유전자의 다양한 표현에 일부가 원인일 수 있습니다. 그럼에도 불구하고, 우리 프로토콜 획득 바이러스 titers은 대부분의 어플 리케이션을위한 충분한보다되며 어떤 경우에는 세포의 과반수의 도입으로 이어질&gt; 세포의 90 %. 원심 분리 기반 기술은 일반적으로 바이러스 입자를 집중하는 데 사용됩니다. 그러나, 원심 분리는 몇 시간을 필요로 전염성 에어로졸을 생성하고, 바이러스 입자의 상당한 손실이 발생할 수 있습니다. 12 대안, CS / PB와 함께 complexation으로이 바이러스 tropism를 변경하지 않고 전달을 향상시키는 데 사용할 수 있습니다. 3이 방법은 (5 분 빠른있다 대략 관찰된 tit​​er를 tripling 동안 (10 ML 바이러스 당 $ 0.03)) 저렴. 특별한 장비 또는 독점적인 시약이 필요하지 않으며 다른 세포 라인의 이전 작업과 동의, CS / PB에 HDFs의 노출 후 최소한의 급성 독성을 관찰하고 있습니다.이 프로토콜 13 한 잠재적인 단점이 몇 가지 미세 표시 폴리머 단지가 고수이다 문화에 몇 일 동안 세포. 우리는이 단지는 세포에 독성 명백히 없지만, 세포 프로세스에 대한 자세한 미묘한 영향을 수있는 가능성을 배제할 수 없습니다. 여기 우리는 안전하고 효과적으로 종양 발생 요인과 인간의 세포를 transducing위한 방법론을 설명합니다. 이러한 접근 방식은 IPS 세포를 포함한 oncogenes 및 줄기 세포 생물학을 공부하면서 연구에 큰 유틸리티의해야합니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> pLenti6/UbC/mSlc7a1 </td><td> Addgene </td><td> 17,224 </td><td> Murine MLV 수용체 </td></tr><tr><td> pMD2.G </td><td> Addgene </td><td> 12,259 </td><td> VSV - G 봉투 </td></tr><tr><td> pCMV - dR8.91 </td><td> D. Trono 연구소 14 </td><td></td><td> 포장 플라스미드 (이에 상응하는 플라스미드 psPax2가 Addgene에서 구할 수 고양이. 넘버 12260) </td></tr><tr><td> pHCMV - EcoEnv </td><td> Addgene </td><td> 15,802 </td><td> Ecotropic 봉투 </td></tr><tr><td> FUGW </td><td> Addgene </td><td> 14,883 </td><td> 플라스미드 GFP 제어 </td></tr><tr><td> OptiMEM </td><td> Invitrogen </td><td> 31,985 </td><td></td></tr><tr><td> Fugene HD </td><td> 로체 </td><td> 04 709 705 001 </td><td></td></tr><tr><td> Hexadimethrine 브로마이드 (Polybrene) </td><td> 시그마 - 알드리치 </td><td> H9268 </td><td></td></tr><tr><td> 상어 연골의 콘드로이틴 황산 나트륨 소금 </td><td> 시그마 - 알드리치 </td><td> C4384 </td><td></td></tr><tr><td> Blasticidin S </td><td> 어부 </td><td> BP 2,647,100 </td><td></td></tr><tr><td> Methylthiazolyldiphenyl - tetrazolium 브로마이드 (MTT) </td><td> 시그마 - 알드리치 </td><td> M2128 </td><td></td></tr><tr><td> Igepal CA - 630 </td><td> 시그마 - 알드리치 </td><td> I3021 </td><td></td></tr></tbody></table>

transduction of human cells with polymer-complexed ecotropic lentivirus for enhanced biosafety

줄기 세포 및 종양 생물학 연구는 종종 실험실 직원에 대한 주요 안전 문제를 제기, 알려진 또는 의심되는 oncogenes와 인간 세포의 바이러스 전달을 필요로합니다. 등 일반적으로 사용되는 VSV - G pseudotype로 Pantropic lentiviruses는, 그들이 아닌 나누어 세포를 포함하여 많은 세포 유형을 transduce 수 있기 때문에 유전자 기능을 연구를위한 유용한 도구입니다. 그러나, 연구자들은 높은 biosafety 수준의 처리 요구 사항 및 안전 문제로 인해 oncogenes을 인코딩 pantropic 바이러스의 생산과 원심 분리를 방지하실 수 있습니다. C - Myc 및 SV40 큰 T 항원을 포함한 여러 적정량 oncogenes은, 유도 pluripotent 줄기 세포 (iPSC)의 생산을 강화하는 것으로 알려져 있습니다. 다른 모든 알려진 iPSC - 유도 유전자 변화 (OCT4, SOX2, KLF4, NANOG, LIN28, 그리고 함수의 p53의 손실)도뿐만 아니라 상대적으로 높은 안전 우려를 제작, 암로 연결되는 링크를 제공하고 있습니다. 이러한 암 관련 바이러스가 세포 프로그래밍 및 pluripotency 공부에 도움이되지만, 그들은 안전하게 사용해야합니다. 이러한 biosafety 문제를 해결하기 위해, 우리는 감소 비용과 편리한 처리에 대한 자세한 중심으로, ecotropic lentivirus와 인간 세포의 형질 도입에 대한 방법을 보여줍니다. 우리는보다 90 % 이상 transduce 충분히 높은 titer와 ecotropic lentivirus을 생산해야 수용체를 표현하는이 방법의 효능을 확인, 바이러스에 노출된 인간의 세포를. Lentivirus은 종종 ultracentrifugation에 의해 집중되어 있으나,이 과정은 몇 시간 걸립니다 인간의 생명 의학 연구에 전염성 에어로졸을 생성하실 수 있습니다. 대안, 바이러스 입자는보다 안전 콘드로이틴 황산 및 polybrene (CS / PB)와 complexation하여 세포에 sedimented 수 있듯이. 이 기술은 안정 무시할 추가 시간과 비용, murine 레트로 바이러스의 수용체를 표현 세포 3 배에 기능 바이러스 titer를 증가시킵니다. 인간 피부 섬유아 세포 (HDFs)의 도입은 maximally되는 고분자 농도가 관심의 대상 세포 유형에 대한 titrated되어야 제안, 이전에 암 세포 라인에 대한보고 최적의 가치보다 약 4 배 낮은 CS / PB 농도를 사용하여 향상된 것입니다. 따라서 새로운 셀 타입 고분자 독성에 대한 분석을 methylthiazolyldiphenyl - tetrazolium 브로마이드 (MTT)의 사용을 설명합니다. 우리는 최소한의 급성 독성을 나타내는, 폴리머 complexation 또는 polybrene (PB, 6 μg / ML)의 표준 복용량을 사용하여 바이러스 전달 후 HDFs의 ​​동등한 가능성을 관찰합니다. 이 프로토콜에서는, 우리는 biosafety 위험을 줄이고 전달 속도를 증가, 인간의 세포에 oncogenes의 overexpression에 대한 ecotropic lentivirus의 사용을 설명합니다. 바이러스 입자 에어로졸 - 성형 원심 분리를 피하는 동안 우리는 또한 전달을 향상시키기 위해 고분자 complexation의 사용을 보여줍니다.

Watch this Scientific Journal Video about Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety at JoVE.com

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

Lentiviruses는 유전자 기능을 탐험을위한 가치있는 연구 도구되지만, 연구진은 알려진 pantropic lentivirus 인코딩이나 의심 oncogenes의 생산을 방지하실 수 있습니다. 대안으로, 우리는 ecotropic 수용체 mSlc7a1을 표현하는 수정된 인간의 세포에 ecotropic lentivirus의 사용을 위해 안전한 프로토콜을 제시한다.

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 ...

transduction-human-cells-with-polymer-complexed-ecotropic-lentivirus

Research

JoVE Journal

Biology

15.0K 조회수.  University of California, Davis. Lentiviruses는 유전자 기능을 탐험을위한 가치있는 연구 도구되지만, 연구진은 알려진 pantropic lentivirus 인코딩이나 의심 oncogenes의 생산을 방지하실 수 있습니다. 대안으로, 우리는 ecotropic 수용체 mSlc7a1을 표현하는 수정된 인간의 세포에 ecotropic lentivirus의 사용을 위해 안전한 프로토콜을 제시한다.

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

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

단백질 Ubiquitination의 감지

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

연구

생물학

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

4 전사 인자와 마우스 배아 섬유아 세포를 프로그래밍하여 유도 Pluripotent 줄기 세포의 생성, Doxycycline는 Lentiviral 전달 시스템 Inducible

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

Lentivirus 제작

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

Stemgent 인간 TF의 Lentivirus와 인간의 섬유아 세포를 프로그래밍하여 유도 Pluripotent 줄기 세포의 생성 세트

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

유전자 인코딩 분자 프로브는 G 단백질 결합 수용체를 연구하는

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

포르말린에서 프로테오믹스 샘플 준비 파라핀 조직에게 고정 및

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

센다이 바이러스와 인간의 체세포의 효율적인 생성 인간 유도 만능 줄기 세포

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

인간 배아 줄기 세포에 표적으로 아연 손가락 핵산 분해 효소 향상된 유전자

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

하나 나노 미터의 정확도와 형광 이미징 (피오나)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

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

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of 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 ...