Name: lentiviral mediated gene silencing in human pseudoislet prepared in low attachment plates
Uploaded: 2019-05-14T13:30:00
Description: Watch this Scientific Journal Video about Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates at JoVE.com

这项工作得到了美国国家卫生研究院(R01-DK090490)和美国糖尿病协会对Y.I.(1-17-IBS-132)的财政支持。J.A.和Y.I.由鹰糖尿病研究中心兄弟会支持。A.B. 得到国家卫生研究院培训补助金(T32NS45549)的支持。作者利用了由NIDDK资助的希望之城综合胰岛分配方案(IIDP)提供的人类胰腺胰岛(2UC4DK098085)。

在开始研究之前,爱荷华大学机构审查委员会作出了人类主题研究的决定,该委员会确定该研究不符合人类学科研究的标准。在开始研究之前,请咨询当地审查委员会,以确定胰岛的来源和计划的研究是否需要事先批准。
注:通常,在96孔超低附着板中形成192个伪胰岛(IEQ)时,需要1,200~1,400个当量(IEQ)的假胰岛,或1,200个伪胰岛大小为500个细胞/伪胰岛。微井文化板。所需的胰?...

<ol>
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J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Li, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+islet+for+improved+performance+by+optimized+reaggregation+in+alginate+gel+beads.">Engineering islet for improved performance by optimized reaggregation in alginate gel beads.</a> Biotechnology and Applied Biochemistry. 64 (3), 400-405 (2017).</li><li>Ramachandran, K., Peng, X., Bokvist, K., Stehno-Bittel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+re-aggregated+human+pancreatic+islets+for+secondary+drug+screening.">Assessment of re-aggregated human pancreatic islets for secondary drug screening.</a> British Journal of Pharmacology. 171 (12), 3010-3022 (2014).</li><li>Hilderink, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+aggregation+of+primary+human+pancreatic+islet+cells+leads+to+glucose-responsive+pseudoislets+comparable+to+native+islets.">Controlled aggregation of primary human pancreatic islet cells leads to glucose-responsive pseudoislets comparable to native islets.</a> Journal of Cellular and Molecular Medicine. 19 (8), 1836-1846 (2015).</li><li>Saunders, D. 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这里给出了一种使用96孔超低附着板或微孔培养板生成由慢病毒转导的人类伪小岛的详细方案。据报道,伪小岛已显示出形态和分泌功能类似于本地人类胰岛,可以在体外进行长时间的体外培养。与在大小上表现出巨大差异的本地人类小岛不同,伪小岛在大小上相对均匀,减少了捐赠者和实验复制体5,11之间的变异。</sup...

功能β细胞质量的丧失是1型和2型糖尿病1的中心病理学。虽然β细胞是胰腺胰岛胰岛素的产生者,但β细胞和非β细胞之间的通信在调节胰岛素分泌2方面起着至关重要的作用。此外,胰高血糖素分泌不良导致糖尿病高血糖3。因此,对胰腺胰岛内细胞的基因表达进行调节,以解决糖尿病胰岛功能障碍发展背后的机制有着浓厚的兴趣。包括转基因小鼠在内的各种方法可用于调节小鼠胰岛的基因表达。然而,人类和小鼠胰岛表现出明显的内向、细胞分布、β与α细胞的比例以及对分泌细胞4的响应。因此,直接评估人类胰岛的基因功能对于理解人类胰腺胰岛的病理生理学具有极其重要的作用。
腺病毒载体是体外转胰腺胰岛应用最广泛的病毒载体,因为非分裂细胞的转导效率很高。然而,腺病毒不能有效地渗透到胰岛的核心,特别是在人类胰岛5,并且在高剂量6细胞毒性。相比之下,慢病毒载体的细胞毒性较小,将外源基因永久地输送到后线细胞的染色体中,使其成为基因治疗7的广泛测试载体。然而,扁豆病毒穿透完整人类胰岛核心的能力也有限,因此需要通过酶消化进行部分分散,以提高转导效率8。完整人类胰岛分散的注意事项是细胞-细胞和细胞基质通信的中断,这损害了胰岛素分泌的动态调节,这对维持人类葡萄糖平衡至关重要9。因此,在人类胰岛模型中评估基因调制对胰岛功能动态调节的影响一直具有挑战性。
众所周知,人类和啮齿类小岛的分散胰岛细胞可自主地重新聚合成称为"伪胰岛"的胰岛状结构。伪胰岛显示β和非β细胞分布类似于原生小岛10,11。此外,经过长期培养后,原生胰岛逐渐失去强大的第一期胰岛素分泌5,10,11,12。然而,与同一文化期5之后的本地小岛相比,伪胰岛在葡萄糖反应下,第一阶段胰岛素分泌的保存效果更好。除了更好地保存胰岛素分泌外,在低附着板11中控制人体小岛细胞的重新聚集,为在将扁病毒载体重新聚集到其中提供了一个机会之窗伪小岛。几项研究已经证明了伪胰岛结合扁病毒介导转导的效用。Caton等人13日报告说,与未感染对照相比,引入表达慢病毒的绿色荧光蛋白(GFP)对胰岛素分泌作用甚微,同时实现大鼠伪胰岛体内GFP的同质表达。他们还通过慢病毒13过度表达康奈辛32、36和43,证明了不同康奈辛对胰岛素分泌的具体作用。用市售的96孔超低附着板制备的人类伪胰岛证明,经慢病毒介导的转录因子SIX3的过度表达可改善静态孵育评估的胰岛素分泌。最近,用96孔超低附着板制备的人类伪胰岛通过慢病毒短发针RNA(shRNA)降低调节葡萄糖素酶,以证明其原理,表明葡萄糖刺激的胰岛素分泌减少,同时KCl刺激胰岛素分泌保存5。研究还表明,人类伪胰岛在基因表达和分泌谱方面与原生胰岛相似,进一步支持了人类伪胰岛对胰岛功能5的调节的效用。虽然没有进行围产,但最近上市的生物工程微孔培养板也报告与慢病毒转导相容,并产生了具有优秀胰岛素的人类假小岛移植后体外和体内分泌11。总体而言,人类假岛的形成与慢病毒转导相结合是研究人类胰岛病理生理学的一种简单而有效的方法,为在人类胰岛中进行机械研究提供了宝贵的工具。
在本报告中,提出了一种利用两个市售平台——96孔超低附着板和微孔培养板——与慢病毒转导的人类伪小岛形成方案。两者都实现了基因表达的有效调制,并创造了与下游评估(包括静态孵育和围气又液)相容的人类伪岛。

<table>
	<tbody>
		<tr>
			<td>Anti-adherence rinsing solution</td>
			<td>Stemcell technologies</td>
			<td>7919</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety cabinet</td>
			<td>Thermo Scientific</td>
			<td>1300 Series Type A2</td>
			<td></td>
		</tr>
		<tr>
			<td>cell strainer, 40 micrometer</td>
			<td>Corning</td>
			<td>431750</td>
			<td></td>
		</tr>
		<tr>
			<td>CMRL-1066</td>
			<td>ThermoFisher</td>
			<td>11530037</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Scientific</td>
			<td>Heracell VIOS 160i</td>
			<td></td>
		</tr>
		<tr>
			<td>conical centrifuge tube, 15 mL</td>
			<td>VWR</td>
			<td>89039-666</td>
			<td></td>
		</tr>
		<tr>
			<td>conical centrifuge tube, 50 mL</td>
			<td>VWR</td>
			<td>89039-658</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>ThermoFisher</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>guanidinium thiocyanate RNA extraction reagent</td>
			<td>ThermoFisher</td>
			<td>15596026</td>
			<td>Trizol</td>
		</tr>
		<tr>
			<td>glutamine</td>
			<td>ThermoFisher</td>
			<td>25030164</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Marien Feld</td>
			<td>Neubauer-Improved Bright line</td>
			<td></td>
		</tr>
		<tr>
			<td>Human serum albumin</td>
			<td>Sigma</td>
			<td>A1653</td>
			<td></td>
		</tr>
		<tr>
			<td>inverted microscope</td>
			<td>Fisher brand</td>
			<td>11-350-119</td>
			<td></td>
		</tr>
		<tr>
			<td>microcentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Microfuge 20</td>
			<td></td>
		</tr>
		<tr>
			<td>microcentrifuge tube, 1.5 mL</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>microwell culture plate</td>
			<td>Stemcell technologies</td>
			<td>34411</td>
			<td>Aggrewell 400, 24 well</td>
		</tr>
		<tr>
			<td>motor-driven pestle</td>
			<td>GAMUT</td>
			<td>#399X644</td>
			<td></td>
		</tr>
		<tr>
			<td>non-tissue culture treated dish, 10 cm</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>ThermoFisher</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 35 mm</td>
			<td>Celltreat</td>
			<td>229638</td>
			<td></td>
		</tr>
		<tr>
			<td>pipette, 5 mL</td>
			<td>DOT Scientific,</td>
			<td>667205B</td>
			<td></td>
		</tr>
		<tr>
			<td>pipette, 8-channel</td>
			<td>VWR</td>
			<td>#613-5253</td>
			<td></td>
		</tr>
		<tr>
			<td>pipette, 10 mL</td>
			<td>VWR</td>
			<td>667210B</td>
			<td></td>
		</tr>
		<tr>
			<td>pipette, P10</td>
			<td>Denville</td>
			<td>UEZ-P-10</td>
			<td></td>
		</tr>
		<tr>
			<td>pipette, P200</td>
			<td>Denville</td>
			<td>UEZ-P-200</td>
			<td></td>
		</tr>
		<tr>
			<td>pipette, P1000</td>
			<td>Denville</td>
			<td>UEZ-P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>proteolytic and collagenolytic enzyme mixture</td>
			<td>Sigma</td>
			<td>A6965</td>
			<td>Accutase</td>
		</tr>
		<tr>
			<td>reagent reservoir, 50 mL</td>
			<td>VWR</td>
			<td>89094-680</td>
			<td></td>
		</tr>
		<tr>
			<td>reversible strainer, 37 micrometer</td>
			<td>Stemcell technologies</td>
			<td>27251</td>
			<td></td>
		</tr>
		<tr>
			<td>swing bucket plate centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra X-14R</td>
			<td></td>
		</tr>
		<tr>
			<td>swing bucket rotor</td>
			<td>Beckman Coulter</td>
			<td>SX4750A</td>
			<td></td>
		</tr>
		<tr>
			<td>tuberculin syringe, 1 mL</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>ultra low attachment microplate, 96 well</td>
			<td>Corning</td>
			<td>4515</td>
			<td></td>
		</tr>
	</tbody>
</table>

lentiviral mediated gene silencing in human pseudoislet prepared in low attachment plates

各种基因工具可用于调节啮齿动物胰腺胰岛的基因,以解剖胰岛基因的功能,用于糖尿病研究。然而,由于物种之间在胰岛结构和功能方面众所周知的差异,从啮齿动物胰岛获得的数据往往不能完全复制或适用于人类胰岛。目前,可用于操纵人类胰岛基因表达的技术非常有限。腺病毒、质粒和寡核苷酸将转基因引入完整胰岛,往往效率低、毒性高。低效率在完整小岛的基因调控研究中尤其成问题,需要高效率。众所周知,酶分散的胰岛细胞在培养中重新聚合,形成称为伪胰岛的球状体。大小控制的人类胰岛细胞的再聚合产生伪胰岛,在长期培养后维持动态第一阶段胰岛素分泌,并提供一个窗口,以有效引入低毒性的慢病毒短发针RNA(shRNA)。这里,描述了使用两个市售的多孔板在慢病毒转导后创建人类伪小岛的详细方案。该协议可以很容易地执行,并允许有效地降低基因和评估使用人类小岛细胞的胰岛素分泌活力。因此,具有慢病毒介导基因调制的人类伪胰岛为评估人类胰岛细胞内的基因功能提供了一个强大而通用的模型。

图 1显示了使用 96 孔超低附着板和微孔培养板生产伪岛的关键步骤。图2a显示了在96孔超低附着板中3 x 103人类胰岛细胞形成伪胰岛期间形态的连续变化。在第1天观察到的单层或松散的细胞团体在第5天至第7天变成具有平滑的圆形边界的固体聚集体(图2a)。在微孔培养板中,固体伪岛的形成通常在4天内...

Watch this Scientific Journal Video about Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates at JoVE.com

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

提出了一种从分散的人类胰岛细胞中产生基因修饰的人类伪胰岛的函数,这种细胞由携带短发核糖核酸(shRNA)的慢病毒转导。该协议利用现成的酶和培养容器,易于执行,并产生适合功能和形态学研究的转基因人类假岛。

伦蒂病毒介导基因在低附着板中制备的人类伪性沉默

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, ...

难以调节人类小岛的基因表达，人类小岛功能的下降幅度也更大，在基因表达调制后也构成重大挑战。通过结合扁病毒中导SHR8转导和形成人类合适的小岛。该协议允许有效降低目标基因的调节，同时在长期培养期间保持小岛功能。首先，轻轻旋转运输瓶，以保持小岛在悬挂。将含有小岛的运输介质转移到 50 毫升锥形离心管中。让管子在生物安全柜中坐15分钟，使小岛安顿到管子的底部。使用毛细管移液器轻轻拆下运输介质，而不会干扰小岛颗粒。用 1% 的人血清白蛋白将 CMRL 介质中的小岛颗粒重新悬浮到每毫升 400 个小岛当量的浓度。将小岛转移到非组织培养皿中，并在 5%的二氧化碳培养箱中以 37 摄氏度进行培养。经过通宵培养后，将人类小岛转移到15毫升锥形离心管中，在摆动的桶转子中以250倍g的离心机进行5分钟。用毛细管移液器吸气介质，而不会干扰小岛颗粒。将颗粒清洗至管中，将10毫升PBS洗净，轻轻混合，以250倍g离心5分钟。离心后，在不干扰小岛颗粒的情况下吸入PBS。将颗粒重新悬浮在预热蛋白解和胶原蛋白酶混合物的0.5毫升中。使用 P1000 移液器混合小岛五次移液器。在37摄氏度下孵育5分钟。孵育后，混合移液器轻轻上下抚摸。检查单细胞云量和薄片或未消化小岛的数量。将 40 微米过滤器放在 35 毫米培养皿中，通过加入 10% 热灭活胎儿牛血清和用 1 毫升注射器柱塞压压，将一毫升 CMRL 介质加入湿化过滤器。转移所有在过滤器顶部的细胞悬浮液。并在一个新的15毫升管收集通过。用0.5毫升的新鲜CMRL介质清洗用于小岛消化的管子，收集剩余的细胞，并通过过滤器进行洗涤。将通道组合在15毫升的管子中。重复一次。要分离留在过滤器上的未消化小岛，请用一毫升注射器柱塞按压过滤器。再次收集通过，用新鲜的普通CMRL 1066介质清洗过滤器，从过滤器和盘子中去除所有剩余的消化小岛。将通道添加到 15 毫升管中。记录细胞悬浮液的总体积，并服用10微升的细胞等分，以计算血细胞计的细胞数。在200倍g下将细胞悬浮液离心5分钟后，在不干扰颗粒的情况下取出介质。要使用96孔超低附着板生产伪小岛，首先计算所需总体积的1倍10至第五细胞每毫升的单细胞悬浮液。然后，用10%的热灭活胎儿血清重新悬浮CMRL培养中的胰岛颗粒，使30微升细胞悬浮液有3000个细胞。接下来，将单细胞悬浮液所需的体积转移到新鲜 15 毫升管中。在含有SH-RNA的扁家病毒每细胞250个转导单元，目标为感兴趣的基因或控制基因。要将细胞悬浮液与病毒混合，移液器用P1000移液器轻轻混合五次。然后，如果使用八通道移液器，将混合细胞转移到50毫升无菌试剂储液罐中。使用八通道移液器或 P200 移液器，将每井的 30 微升与扁病毒混合到每个井中。接下来，在室温下以270倍g的270倍g将96井板离心7分钟。在37摄氏度的高温下，在一夜之间在一个加湿的5%二氧化碳孵化器中。孵育后，从培养箱中取出96井板，放在生物安全柜中。每个小岛100微升的10%高FBSCMRL进入井，以防止小岛干燥，并继续培养一个星期。每孔CMRL介质的移液器100微升，用10%的热灭活胎儿牛血清从储液罐到伪胰岛，移液器上下两到三次轻轻地在井中抬起小岛。然后，吸气的介质在井中含有一个伪岛，并弹出到10厘米的培养皿。在光学显微镜下检查板，以确保完全去除所有伪小岛，然后进行下游实验。要使用 24 孔微井培养板生产伪小岛，首先添加 500 微升每孔的抗粘附冲洗溶液。在摆动铲斗板离心机中以 1300 倍 g 离心，5 分钟。然后，在显微镜下观察板，以确保从微孔中去除气泡。在生物安全柜中，从井中吸出防粘剂冲洗溶液。用两毫升暖普通CMRL 1066介质冲洗每口井一次。然后，吸气平原CMRL 1066介质，并添加0.5毫升每井温暖的CMRL与10%的热灭活胎儿牛血清到每个精心规划使用。确定每一个井所需的细胞总数。用10%的热灭活胎儿血清在无菌的1.5毫升管中，用0.8毫升CMRL培养的单个细胞重新悬浮。为了制造一口由扁病毒转导的伪岛井，每个细胞增加125个转导单元，使病毒的体积保持在0.2毫升以下。在37摄氏度下孵育细胞和病毒混合物，偶尔温和混合一小时，使细胞与病毒接触，然后凝结细胞。一小时后，通过添加CMRL培养器和10%热灭活胎儿牛血清，将胰岛细胞和病毒混合物的总体积调整为1毫升。如果细胞在一小时后形成团块，通过温和和快速移液两到三次分散到单个细胞悬浮液中。将电池悬浮液转移到 24 井微井培养板的一个井中。紧接着移液，在室温下以100倍g离心3分钟，将细胞捕获到所有微孔中。在显微镜下观察，验证细胞在所有微孔中均匀分布。将小岛单细胞悬浮液加入井中后，立即将微井板离心至关重要。在96孔超低附着板中创建的3000个人类小岛细胞的伪小岛形态的连续变化表明，单层或松散的细胞团在一周内以平滑的圆边界变成固体聚集体。相比之下，在微井培养板中，细胞和伪小岛的形成通常仅在4天内可见。伪小岛的统一大小减少了测试组内的变异，并允许静态孵育，每个测量只使用五个伪小岛。Perry融合胰岛素反应分析显示，人类伪不对岛在培养七天后，对葡萄糖保持了强大的第一阶段胰岛素分泌。将针对人类脂肪甘油三酯脂肪酶或五个基因的扁病毒引入单个细胞悬浮液中，确保小岛细胞高效、均匀的转导，并实现对基因的高效低调节。重要的是要记住，将人类小岛分散到单个细胞中所用的时间受许多因素的影响，如：大小、分布和供体特征。因此，在消化过程中密切注意人类小岛消失是十分重要的。在整个协议中，轻轻处理单细胞悬浮液以避免细胞丢失非常重要。正在创建的人类伪小岛可用于许多应用，以确定基因调制的效果，如：西方的布洛特，组织学研究和氧气消耗率的测量。该协议允许使用常见的实验室软件和简单的技术，评估目标基因在调节小岛健康和人类小岛功能方面的低值。使用人类小岛可能需要事先获得当地审查委员会的批准。在开始研究之前，请咨询当地审查委员会。伦蒂病毒被归类为生物安全二级。在开始使用扁病毒之前，请与生物安全委员会联系。

Research

JoVE Journal

Genetics

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

科研

遗传学

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

低丰度胚胎标本的染色质沉淀 (芯片) 协议

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...