这项工作的部分原因是由辛辛那提儿童研究基金会 (CCRF) 为 M.N. 和创新核心赠款提供资金, 该基金由国家卫生研究院促进翻译科学中心资助。奖项编号 UL1 TR001425。 内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。

有关汇总协议的说明, 请参见图 1 。
1. 杆状病毒质粒 DNA 的纯化
<ol><li>用抗生素在200毫升的 LB 汤中生长含有杆状病毒质粒 DNA (bacmid) 14 的细菌培养;卡那霉素 (50 µg/毫升), 四环素 (10 µg/毫升), 和庆大霉素 (7 µg/毫升), 16 h 在37°c 在一个轨道振动筛设置在 300 rpm。</li>
	<li>使用标准碱性裂解协议15从细菌培养中纯化 bacmid DNA, 并将 bacmid 溶解在 TE 缓冲器中。</li>
</ol>2. 杆状病毒的生产
<ol><li>培养 Sf9 细胞的轨道振动筛孵化器在 135 rpm 和28°c 的聚碳酸酯锥形瓶含有无血清昆虫培养介质1,2,3。 注意:如果 Sf9 细胞从冷冻的库存解冻, 允许至少两个星期的细胞恢复和进入指数阶段的增长。</li>
	<li>用台盼蓝染色 hemocytometer, 计数在生长指数阶段 Sf9 细胞。种子 1 x 106可行的 Sf9 细胞每井在6井组织培养处理的板材在2毫升培养基中。允许单元格在孵化器中的28摄氏度处附加1小时。</li>
	<li>用1毫升无血清 unsupplemented 的昆虫培养基取代生长培养基。</li>
	<li>利用昆虫细胞转染试剂将 bacmid DNA 染到 Sf9 细胞中。<ol>
<li>将8µL 昆虫细胞转染试剂与100µL 的宽限期昆虫培养基混合使用。</li>
		<li>将2µg 的 bacmid DNA 稀释成 unsupplemented 昆虫培养基的100µL, 并通过涡流轻轻混合。</li>
		<li>将稀释后的 DNA 与稀释后的昆虫细胞转染试剂结合, 轻轻混合。室温下孵育15至30分钟。</li>
		<li>将 DNA 脂质混合物滴在细胞上。孵化在28°c 在一个孵化器大约5小时。</li>
	</ol>
</li>
	<li>从细胞中取出转染的混合物, 用2毫升的 PBS 冲洗细胞。</li>
	<li>添加2毫升的昆虫培养基, 并继续培养在28摄氏度的孵化器, 而不改变媒体。 注意:如果 bacmid 含有荧光蛋白, 其表达可以在48小时后转染细胞中检测到。杆状病毒由转染的 Sf9 细胞产生, 并分泌到随后感染 untransfected 细胞的培养基中。整个细胞群在5天内就会感染杆状病毒, 并显示病毒感染的迹象, 也称为病变效应。细胞培养中包括细胞直径的增加, 胞浆中的空泡/颗粒, 细胞生长的停止和死或裂解细胞的存在。然而, 如果转染或感染的效率不是最佳, 文化可能不会显示明显的病变效果。病变效应可以用 200-400X 放大的倒置相显微镜进行可视化。用 anti-gp64 抗体11染色可检测杆状病毒感染细胞。</li>
	<li>在转染和标签为 P1病毒储存后5天收获杆状病毒上清。</li>
	<li>储存的杆状病毒上清, 这是轻敏感, 在黑暗中与0.5% 牛血清白蛋白 (BSA) 在 PBS。贮存在4°c 为短期 (最多90天) 或在-80 摄氏度长期。</li>
	<li>种子 6 x 106 Sf9 细胞在 10 cm 组织培养处理的板材在10毫升昆虫培养基。将1毫升的最初杆状病毒上清 (P1) 添加到细胞中。</li>
	<li>收获杆状病毒上清 (P2) 在 5th天后感染。</li>
	<li>种子50毫升的 Sf9 细胞 (3 x 106细胞/毫升) 的悬浮培养在一个250毫升聚碳酸酯锥形瓶, 并把瓶子在一个轨道振动筛孵化器。将2毫升的 P2库存添加到单元格中, 并在 135 rpm 处晃动, 同时保持28°c 的恒定温度。 注意:感染后三或四天, 整个 Sf9 细胞将显示病变效应, 这是高效杆状病毒产生的征兆。</li>
	<li>依次放大杆状病毒上清液直到获得所需的体积 (p3,p4,...) 通过增加10到50倍的细胞培养量在随后的每一次感染。 注意:p3是 3rd一轮感染, 其中500毫升至 1 L 的 Sf9 细胞培养可感染10至20毫升 P2杆状病毒上清;p4是 4th的一轮感染, 其中5至 10 L 的 Sf9 细胞培养可感染100至200毫升 P3杆状病毒上清, 等等。通常, 在无血清昆虫培养基中, Sf9 细胞的种子为 3 x 106细胞/毫升。</li>
	<li>离心的杆状病毒上清 1000 x g 30 分钟在4°c 去除细胞碎片和治疗杆状病毒上清与50单位/毫升核酸酶 (250 单位/µL 股票) 2 摄氏度, 以消化基因组 DNA 和 RNA。通过0.45 微米的过滤单元过滤上清液。</li>
</ol>3. 色谱系统的制备
<ol><li>用无菌水、1氮氢氧化钠、水和洗涤缓冲器 (20 毫米磷酸钠缓冲液, 含150毫米氯化钠, pH 7.0), 以50毫升/分钟的线性流速, 依次冲洗样品和缓冲线, 制备色谱系统。</li>
	<li>准备一个肝素50µm 柱 (10 x 10 厘米, 7.9 毫升的柱容积 (CV)) 按顺序运行的柱清洁缓冲 (5 cv 无菌20毫米磷酸钠缓冲液含2米氯化钠, pH 7.0) 和 5 CV 洗涤缓冲器在线性流量的7毫升/分钟。</li>
</ol>4. 杆状病毒载体的纯化
<ol><li>建立色谱系统如下:<ol>
<li>使用样品装货入口口岸装载杆状病毒上清。</li>
		<li>使用缓冲入口端口 A1 加载洗涤缓冲器。准备一瓶至少500毫升的洗涤缓冲器, 并将洗涤缓冲线放进瓶子里。</li>
		<li>使用缓冲入口端口 A2, 用于装载洗脱缓冲器 (20 毫米磷酸钠, 1.5 米氯化钠, pH 值 8.0)。准备一瓶至少500毫升洗脱缓冲器, 并将洗脱缓冲线放进瓶子里。</li>
		<li>将50毫升锥形管插入到分数收集器中, 收集柱通过杆状病毒上清, 洗涤缓冲器和洗脱杆状病毒。</li>
	</ol>
</li>
	<li>平衡五7.9 毫升的洗涤缓冲器 (40 毫升) 的肝素柱, 其线性流速为7.0 毫升/分。</li>
	<li>用色谱系统的采样泵 (进样口) 将250毫升的杆状病毒上清液载入肝素柱上, 其线性流速为2.0 毫升/分。</li>
	<li>运行 10 CVs (80 毫升) 的洗涤缓冲通过肝素柱在线性流量2.0 毫升/分钟, 直到紫外线 (紫外线) 吸收曲线 (280 nm) 已经返回到基线, 并变得稳定。</li>
	<li>洗脱7.9 毫升肝素柱上的杆状病毒粒子, 其 5 CVs (40 毫升) 的洗脱缓冲器的线性流速为4.0 毫升/分. 观察当杆状病毒粒子与肝素柱分离时, 色谱中蛋白质的尖锐洗脱峰。</li>
	<li>洗脱后, 立即稀释洗脱杆状病毒上清10倍, 使用20毫米磷酸钠缓冲液, 以防止在随后的离心过程中, 杆状病毒颗粒从渗透冲击中失活。</li>
	<li>存储100µL 的每个分数, 包括列流-通过收集在加载的杆状病毒上清, 洗涤缓冲区, 和洗脱液。感染这些杆状病毒分数 HT1080, 以评估纯化过程 (见第 6)。</li>
	<li>用列清理缓冲区和洗涤缓冲区清理列。最后, 用 5 CV 无菌20% 乙醇在水中以7.0 毫升/分钟的线性流速冲洗柱, 并贮存在4摄氏度。</li>
	<li>用水、1氢氧化钠、再用水和20% 乙醇在水中以50.0 毫升/分钟的线性流速清洗色谱系统, 并将系统储存在20% 乙醇中。</li>
</ol>5. 杆状病毒的浓度
<ol><li>使用高压釜预杀菌离心管。添加一个相同数量的杆状病毒上清到每个 ultracentrifuge 管在组织培养罩。通过平衡测量管的重量, 并调整 ultracentrifuge 管的含量与无菌 PBS 的重量。</li>
	<li>将每个 ultracentrifuge 管放置在 SW 32 钛转子的对立桶中。</li>
	<li>在4摄氏度时, 以 8万 x g 运行 ultracentrifuge 90 分钟. </li>
	<li>在组织培养罩中打开 ultracentrifuge 管, 灌装在不去除杆状病毒颗粒的情况下吸入液体。</li>
	<li>并用重悬吹打的200µL PBS, 用 0.5% (w/卷) BSA, 通过有力地将每根管子的颗粒向上和向下。</li>
	<li>将浓缩的杆状病毒转移到 cryovials (每瓶50到100µL), 并存储在-80 摄氏度。</li>
</ol>6. 杆状病毒滴定法
<ol><li>感染前的前一天, 用台盼蓝染色细胞后使用 hemocytometer 计数 HT1080 细胞。种子 1 x 105 HT1080 细胞每井在6井板中的2毫升的 Dulbecco 的改良鹰的培养基 (DMEM) 含有10% 胎牛血清 (血清)。种子几个额外的水井能够确定在感染时存在的细胞的确切数量。在孵化器的37°c 和 5% CO2中培养细胞。 注意:HT1080 细胞用于滴定, 因为它们表达较高水平的硫酸硫酸与其他细胞线16。由于 HT1080 细胞是黏附的, 滴定比使用传统的 Sf9-based 滴定更简单, 更少的时间消耗。</li>
	<li>在感染日, 通过对水井中的细胞计数, 确定每个井的细胞数。通过添加稀释后的原杆状病毒, 在柱纯化之前被搁置, 并通过从柱的流动, 洗涤和洗脱分数的样本来感染细胞。对每个样品, 根据传染性杆状病毒粒子的经验, 确定稀释量和体积, 并对每一个井进行三种感染。</li>
	<li>感染后两天后, 分析细胞对基因表达的兴趣。如果细胞表达荧光蛋白 (例如GFP)17, 则可以使用流式分析仪进行分析。</li>
	<li>根据在感染时存在的细胞数、稀释因子和细胞中荧光蛋白的百分比, 计算出的滴度 (每毫升感染单位): (感染期间细胞数 x 荧光蛋白的百分比阳性细胞 x 稀释因子)/杆状病毒的体积。 注意:例如, 如果感染时每井细胞总数为 1.2 x 105, 荧光蛋白的百分比为 5%, 稀释因子为 100, 稀释杆状病毒的体积增加10µL, 效价为 6 x 107传染性单位 (IU)。毫升.</li>
</ol>

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作者声明没有利益冲突。

本文介绍了在悬浮培养中 Sf9 细胞中杆状病毒的产生和用肝素亲和层析纯化杆状病毒的方法。该协议中使用的参数最大限度地提高了感染杆状病毒的产量, 最大限度地减少了其灭活。此处提供的协议显示, 与其他9实现的恢复相比, 杆状病毒粒子的恢复明显改善。
由于广泛的寄主范围和组织取向, 在中枢神经系统、肝、眼、卵巢、前列腺和睾丸中进行了多项研究...

杆状病毒主要用于生产重组蛋白和疫苗的鳞翅目斜纹 fugiperda (Sf) 9 昆虫细胞使用重组杆状使用杆状 multicapsid 核多角体病毒 (AcMNPV)1,2,3,4. 最近, 它正在成为基因治疗应用5的一个有希望的载体。众所周知, 它具有广泛的宿主和组织取向, 感染静止和增殖细胞, 是非致病性的, 并没有集成到主机染色体4,5,6。此外, 杆状病毒可以在无血清悬浮培养, 可伸缩性, 并允许为未来的临床生产关闭系统处理1。
杆状病毒粒子的纯度对于实现有效的转导非常重要, 同时尽量减少细胞毒性7,8,9。杆状病毒可以通过离心或切向流动过滤 (TFF) 来集中, 对其传染性有有限的影响。然而, 这些程序不仅集中了病毒微粒, 而且还有来自 Sf9 文化的细胞碎片和蛋白质, 这可能是有毒的体外(个人观察), 在使用体内时可能诱发炎症或免疫应答。为了避免这种情况, 特别是在使用高度集中的病毒储存时, 感染性杆状病菌需要纯化, 并与污染微粒分离。
报告了几种用于纯化和浓缩杆状病毒载体的方法10,11,12。在现有的方法中, 肝素亲和色谱允许单步高水平的纯化与低浓度的污染蛋白12。该方法是基于硫酸硫酸根的识别作为杆状病毒13,14的受体。在将 Sf9 细胞上移到杆状病毒的柱上并结合后, 该柱可以用生理 (等渗) 缓冲器冲洗, 以去除未绑定或松散的污染微粒。由于对肝素的结合是可逆的, 杆状病毒颗粒可以洗脱高盐缓冲, 这是立即稀释到生理 (等渗) 盐浓度, 以防止通过渗透冲击 12.此外, 杆状病毒的生产, 以及从色谱柱上捕获和洗脱, 可以使用一个封闭系统的过程, 这是符合当前良好的生产实践 (cGMP)。
在这里, 我们提供了一个详细的协议, 以制造, 纯化和浓缩的传染性杆状病毒使用亲和层析和离心。简要地, 我们生产杆状病毒通过转染 Sf9 细胞与杆状病毒质粒 DNA 在黏附培养和进一步扩大传染性杆状病毒在无血清悬浮培养。我们用肝素亲和层析纯化杆状病毒, 并以离心为最后一步, 高度集中载体进行基因治疗应用。

<table border="0" cellpadding="0" cellspacing="0" style="width:784px;" width="784">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="16">
			<td height="16" style="height:16px;width:257px;">Akta Avant 150</td>
			<td style="width:204px;">GE Healthcare</td>
			<td style="width:156px;">28976337</td>
			<td style="width:167px;">Chromatography system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:257px;">POROS Heparin 50 &#181;m Column</td>
			<td>ThermoFisher Scientific</td>
			<td>4333414</td>
			<td>Heparin column</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Ultracentrifuge</td>
			<td>Beckman-Coulter</td>
			<td>Non-catalog item</td>
			<td>Concentrates virus at high-speed</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Polyallomer Ultracentrifuge tube</td>
			<td>Beckman-Coulter</td>
			<td>326823</td>
			<td>Concentrates virus at high-speed</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">MaxQ 8000 orbital shaker incubator&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>Non-catalog item</td>
			<td>Shaker for suspension culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 mL Erlenmeyer flasks</td>
			<td>ThermoFisher Scientific</td>
			<td>238071</td>
			<td>Flask for suspension culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 L Erlenmeyer flasks</td>
			<td>ThermoFisher Scientific</td>
			<td>238072</td>
			<td>Flask for suspension culture</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Microscope (Olympus CKX41)</td>
			<td>Olympus</td>
			<td>Non-catalog item</td>
			<td>Cell monitoring and counting&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Table top centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75253839/433607</td>
			<td>For clarification of Baculovirus supernatant</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">50 ml Conical tube</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-49A</td>
			<td>For collection of Baculovirus supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plate</td>
			<td>ThermoFisher Scientific</td>
			<td>07-200-80</td>
			<td>Tissue culture treated plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10-cm plate</td>
			<td>ThermoFisher Scientific</td>
			<td>08-772E</td>
			<td>Tissue culture treated plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Stericup-HV, 0.45&#160;&#181;m, PVDF</td>
			<td>EMD-Millipore</td>
			<td>SCHVU05RE</td>
			<td>Filtration unit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15160-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracycline</td>
			<td>Sigma-Aldrich</td>
			<td>T7660</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Gentamycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bac-to-Bac Vector Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>10360-014</td>
			<td>Baculovirus expression system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DH10B-T1R&#160; Competent cell</td>
			<td>ThermoFisher Scientific</td>
			<td>12331-013</td>
			<td>Competent cell for bacmid</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">TE buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>10 mM Tris-HCl, 1 mM EDTA, pH 8.0.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid Maxiprep kit</td>
			<td>ThermoFisher Scientific</td>
			<td>K2100-06</td>
			<td>For bacmid purification</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sf9 Cells</td>
			<td>ThermoFisher Scientific</td>
			<td>11496-015</td>
			<td>Insect cells</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Grace&#8217;s Insect Cell Culture Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11605-094</td>
			<td>Transfection medium</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>20012227</td>
			<td>Washing cells, diluting samples</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:257px;">HyClone SFX-Insect cell media</td>
			<td>GE Healthcare</td>
			<td>SH30278.02</td>
			<td>Serum-free insect cell growth medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Benzonase Nuclease</td>
			<td>Sigma-Aldrich</td>
			<td>E1014</td>
			<td>Enzyme to degrade DNA and RNA</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Baculovirus plasmid (bacmid) DNA</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>Bacmid for Baculovirus Production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellfectin II&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>10362</td>
			<td>Transfection reagent for insect cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4737</td>
			<td>Stabilizes Baculovirus</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cryovial</td>
			<td>Thomas Scientific</td>
			<td>1222C24</td>
			<td>For storage of Baculovirus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HT1080 cell line</td>
			<td>ATCC</td>
			<td style="width:156px;">CCL-121</td>
			<td>Fibroblast cell line</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
			<td>Growth media for cell lines</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Wash buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>20 mmol/l phosphate buffer containing 150 mmol/l sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elution buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>20 mmol/l phosphate buffer containing 1.5 mol/l sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Column cleaning buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>20 mmol/l phosphate buffer containing 2.0 mol/l sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile water</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>For Akta Avant cleaning</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>1.09137</td>
			<td>For Akta Avant cleaning</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7073</td>
			<td>For Akta Avant cleaning</td>
		</tr>
	</tbody>
</table>

production and purification of baculovirus for gene therapy application

杆状病毒传统上被用于生产重组蛋白和疫苗。然而, 最近, 杆状病毒正逐渐成为基因治疗应用的一个有希望的载体。在这里, 杆状病毒是由 Sf9 细胞黏附培养的杆状病毒质粒 DNA (bacmid) 的瞬态转染而产生的。杆状病毒随后在无血清悬浮培养的 Sf9 细胞中展开, 直到获得所需的体积。然后用肝素亲和层析法从培养上清液中纯化。由于肝素对杆状病毒包络糖蛋白的亲和性, 在上清液中将杆状病毒颗粒结合在一起的肝素柱上上清。该列用缓冲区冲洗, 以去除污染物, 而杆状病毒则是用高盐缓冲柱洗脱的。洗脱液被稀释为一个等渗盐浓度, 杆状病毒粒子进一步集中使用离心。使用这种方法, 杆状病毒可以集中到500倍, 25% 的传染性微粒的恢复。虽然这里描述的协议表明了从培养到1升的杆状病毒的生产和纯化, 该方法可以扩大在封闭系统悬浮培养, 以产生一个临床级的载体, 用于基因治疗的应用。

所提供的协议在流程图中 (图 1)。步骤包括对 bacmid DNA 的 Sf9 细胞进行瞬态转染, 在板的黏附培养中产生杆状病毒, 随后在无血清悬浮培养中扩增, 核酸酶处理和离心和过滤澄清,用肝素亲和色谱法纯化后, 离心浓度。
解冻后, Sf9 细胞需要至少两个星期的时间来恢复和进入指数阶段的增长。积极生长的 Sf9 细?...

Watch this Scientific Journal Video about Production and Purification of Baculovirus for Gene Therapy Application at JoVE.com

Production and Purification of Baculovirus for Gene Therapy Application

杆状病毒基因治疗应用的生产与纯化

在该协议中, 杆状病毒是通过瞬态转染杆状病毒质粒进入 Sf9 细胞并在无血清悬浮培养中扩增而产生的。用肝素亲和层析法纯化上清液, 进一步浓缩离心。该协议对用于基因治疗的杆状病毒的制备和纯化具有重要意义。

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

production-purification-baculovirus-for-gene-therapy

Research

JoVE Journal

Genetics

14.6K 次观看.  Cincinnati Children's Hospital Medical Center. 在该协议中, 杆状病毒是通过瞬态转染杆状病毒质粒进入 Sf9 细胞并在无血清悬浮培养中扩增而产生的。用肝素亲和层析法纯化上清液, 进一步浓缩离心。该协议对用于基因治疗的杆状病毒的制备和纯化具有重要意义。

视频: Production and Purification of Baculovirus for Gene Therapy Application

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70&#916;. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

可再生生物燃料及生化生产的非常规酵母基因工程

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

科研

遗传学

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

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 and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

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 ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

斑马斑马模型系统中 CRISPR/Cas9-generated 基因挖空的高效生产与鉴定

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h.

非人类灵长类动物造血干细胞和祖细胞的制备及基因修饰

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

心脏细胞治疗中干细胞 dna 完整性的评估

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

remtoe 控制系统在内源基因表达调控中的应用

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

转化、基因组编辑和拟合氮的热带大麻树帕拉斯波尼亚安德森

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated miRNAs rather than the action of a single miRNA. The characterization of these specific miRNAs modules is a fundamental step in maximizing their use in therapy. This is extremely relevant because their combinatorial attributes can be practically exploited. Described here is a method to define a specific miRNA signature relevant to the control of oncogenic chromatin repressors in glioblastoma. The approach first defines a general group of miRNAs that are deregulated in tumors in comparison to normal tissue. The analysis is further refined by differential culture conditions, underscoring a subgroup of miRNAs that are co-expressed simultaneously during specific cellular states. Finally, the miRNAs that satisfy these filters are combined into an artificial polycistronic transgenes, which is based on a scaffold of naturally existing miRNA clusters genes, then used for overexpression of these miRNA modules into receiving cells.

Described here is a protocol for characterizing modules of biologically synergistic miRNAs and their assembly into short transgenes, which allows simultaneous overexpression for gene therapy applications.

胶质母细胞瘤中功能相关miRNA的特性及其工程在基因治疗的人工簇中

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated ...

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

使用冷冻胚胎进行转基因小鼠高效生产

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...