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

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JoVE Journal

Biology

15.1K 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

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

신경의 분석을위한 성인 마우스 V-SVZ에서 세포의 안정적이고 효율적인 유전자 변형 줄기 세포 자율 및 비 자치 효과

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

연구

발생학

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 ...

면역학 및 감염병학

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene switches requires the transfer of two therapeutic expression units concurrently into a single cell, the practical application of the system for gene therapy is limited. To simplify the transgene expression system, we generated a gene switch designated as pEUI(+) encompassing a complete set of transgene expression modules in a single vector. Comprising of the GAL4 DNA-binding domain and modified EcR (GvEcR), a minimal VP16 activation domain fused with a GAL4 DNA-binding domain, as well as a modified Drosophila ecdysone receptor (EcR), the newly developed singular gene switch is highly responsive to the administration of a chemical inducer in a time- and dosage-dependent manner. The pEUI(+) vector is a potentially powerful tool for improving the control of transgene expression in both biological research and pre-clinical studies. Here, we present a detailed protocol for modulation of a transient and stable transgene expression using pEUI(+) vector by the treatment of tebufenozide (Teb). Additionally, we share important guidelines for the use of Teb as a chemical inducer.

Here, we present a protocol for the modulation of transgene expression using pEUI(+) singular gene switch by tebufenozide treatment.

Ecdysone 수용 체 기반 단일 유전자 스위치 견고성, 가역, 및 무시할 수 시키는 Transgene의 의도적인 표현에 대 한

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

유전학

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

암 관련 유전자 기능을 연구하기 DNA를 벡터 기반의 RNA 간섭

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

의학

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

기본 인간 T 세포 piggyBac Transposon 시스템 변경

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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 ...

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

종양유전자 유래 전사-복제 충돌을 연구하기 위한 근접 결찰 분석

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

암 연구학

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 ...

생물학

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.

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

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

효율적인 체 외에서 이항 메서드인 Transcriptionally 규제 자 아름다움 시스템 통합 결함이 Lentiviral 벡터에 포장 하 여

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Murine 골 수 유래 수지상 세포와 대 식 세포에 형광 성 융해 단백질의 표정

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...