作者要感谢由于篇幅限制而未在这项工作中引用的对该领域的许多贡献，以及提供这一机会的资助机构。作者感谢加拿大自然科学与工程研究委员会（NSERC）和加拿大卫生研究院（CIHR）的资助，他们支持了这项工作。K.M. 是 NSERC 的 CGS-M 奖学金和不列颠哥伦比亚大学的 Killam 博士奖学金的获得者。NS是迈克尔·史密斯健康研究BC学者奖的获得者。

1. mPSC培养试剂的制备
<ol><li>准备 N2 补充剂。<ol><li>在非无菌条件下，在化学安全通风橱中制备以下储备溶液（步骤1.1.1.1）。将每种化学品的固体粉末加入预先称量的 50 mL 锥形管中。加入粉末后称量每管，加入适量溶剂，达到以下浓度。对于一批培养基，请准备列出的最小量。 注意：以下试剂是危险的，应按照当地的化学品安全指南进行处理。处理时确保适当的个人防护装备，并且仅在化学通风橱中使用这些化学品的固体粉末形式，以防止吸入。<ol><li>0.05 mL 亚硒酸钠在 0.518 mg/mL 的水溶液中，0.5 mL 腐胺在 160 mg/mL 的水溶液中，0.165 mL 黄体酮在 0.6 mg/mL 的 100% 乙醇溶液中（见 材料表）。</li>
			<li>将溶液储存在-20°C长达2年。</li>
		</ol></li>
		<li>在生物安全柜 （BSC） 中，将以下内容添加到 58.035 mL DMEM-F12 中。<ol><li>0.05 mL 0.518 mg/mL 亚硒酸钠溶液、0.5 mL 160 mg/mL 腐胺溶液、0.165 mL 0.6 mg/mL 黄体酮溶液。</li>
			<li>过滤器 用 0.22 μm 过滤器对上述混合物进行灭菌。</li>
		</ol></li>
		<li>仍在 BSC 中，制备 100 mg/mL 脱脂转铁蛋白溶液。<ol><li>将 5 mL DMEM-F12 加入 500 mg 载脂转铁蛋白（参见 材料表）。慢慢重新悬浮并清洗容器，避免气泡。</li>
			<li>用 5 mL 重悬并尽可能多地去除后，将其添加到亚硒酸盐/腐胺/黄体酮溶液中。取更多以前的溶液并清洗脱脂蛋白瓶，尽可能多地取出。</li>
		</ol></li>
		<li>向溶液中加入 5 mL 7.5% BSA 馏分 V（参见 材料表）。</li>
		<li>每管混合并等分 6.875 mL。将等分试样储存在-80°C长达1年。</li>
		<li>使用上述 N2 等分试样制备 N2B27 培养基时，解冻所需的等分试样，并向 6.875 N2 中加入 3.125 mL 4 mg/mL 胰岛素溶液（参见 材料表），以获得 10 mL 100x N2。</li>
	</ol></li>
	<li>准备 N2B27 基础培养基。<ol><li>将 N2 和 B27 补充剂在 4 °C 冰箱中解冻数小时或过夜。</li>
		<li>在 BSC 中，将 484.5 mL 的 DMEM-F12 和 484.5 mL 的 Neurobasal 培养基（参见 材料表）混合在无菌的 1 L 瓶中。</li>
		<li>向培养基中加入 10 mL B27 和 10 mL N2 添加剂。</li>
		<li>加入 1 mL 55 mM β-巯基乙醇（参见 材料表）。加入 10 mL 100x 谷氨酰胺。通过旋转瓶子剧烈混合。</li>
		<li>每等分试样（通常为100mL）等分所需体积，并在-80°C下储存长达1年。在4°C下解冻后储存使用长达3周。</li>
	</ol></li>
	<li>准备 NBiL 介质。<ol><li>在 4 °C 冰箱中从 -80 °C 解冻 100 mL 等分试样的 N2B27 基础培养基。</li>
		<li>在平衡计分卡中，将 LIF（参见 材料表）添加到 10 μg/L 的终浓度。</li>
		<li>加入CHIR99021（3μM最终）和PD0325901（1μM最终）（参见 材料表）。用 50 mL 血清移液管充分混合。在4°C下储存长达2周。</li>
	</ol></li>
	<li>制备0.2%明胶溶液。<ol><li>将 500 mL 双蒸馏 H2O 转移到 1 L 玻璃瓶中。</li>
		<li>量出 1 克明胶（见 材料表）并将其加入水中。摇晃瓶子混合，直到大部分溶解。</li>
		<li>将明胶和水混合物在15psi，121°C下高压灭菌35分钟。冷却后，在 BSC 中用 0.22 μm 过滤器对其进行过滤灭菌。储存在4°C。</li>
	</ol></li>
	<li>准备洗涤介质。<ol><li>在 BSC 中，每 10 mL DMEM-F12 混合 160 μL 7.5% BSA。</li>
		<li>扩大上述规模并大量制备，以便将来使用（例如，8 mL 7.5% BSA 至 500 mL DMEM-F12）。储存在4°C。</li>
	</ol></li>
</ol>2. mPSC多转染试剂的制备
注意：为 24 孔板的单个孔提供了以下值。可以相应地缩放值。所有DNA/质粒的序列详见 补充文件1。
<ol><li>计算每次治疗所需的DNA溶液体积。<ol><li>每个孔/条件制备 500 ng DNA。<ol><li>如果用 100 ng/μL 的 DNA 溶液转染单个质粒，则用 5 μL DNA 制备一个试管。</li>
			<li>如果以100 ng/μL的速率转染两个质粒，则准备两个试管，每个试管中含有2.5μL，每个质粒一个。</li>
		</ol></li>
	</ol></li>
	<li>在 BSC 中，执行以下操作：对于每 500 ng 转染处理（参见 材料表），将 25 μL OptiMEM 等分试样放入 1.5 mL 试管中。<ol><li>相应地缩放 - 在上面的示例中，准备两个试管，每个试管含有 250 ng DNA （2.5 μL） 和 12.5 μL OptiMEM。</li>
	</ol></li>
	<li>将该治疗所需的 DNA 体积添加到试管中的 OptiMEM 中。</li>
	<li>对于每个含有 500 ng DNA 和 OptiMEM 的试管，使用另外 25 μL OptiMEM 和 1 μL 阳离子脂质转染试剂创建一个匹配的试管。<ol><li>同样，必须相应地缩放这些值。对于上述示例，准备两个试管，每个试管含有 12.5 μL OptiMEM 和 0.5 μL 转染试剂。 注意：缩放值时，请使用添加的 DNA 质量，而不是该数量的 DNA 所需的体积。转染试剂和 DNA 的反应可以按比例缩放（例如，如果要用相同的 DNA 转染 5 个孔）。保持试剂的比例相同，但相应地缩放（例如，1000 ng DNA：50 μL OptiMEM：2 μL 转染试剂：50 μL OptiMEM）。</li>
	</ol></li>
</ol>3. 用于转染的mPSCs的制备
注意：对于反向转染，在添加转染试剂之前，准备培养容器并直接传代 mPSC。对于正向转染，在转染前 12-18 小时传代并铺板细胞，以使细胞粘附在平板上。
<ol><li>准备0.2%明胶包被的细胞培养容器。<ol><li>计划转染所需的孔数（每个处理/组一个 24 孔板的孔数）。</li>
		<li>将 200 μL 0.2% 明胶溶液加入 24 孔板中的每个所需孔中。</li>
		<li>轻轻摇晃板以均匀散布溶液，确保它覆盖整个井底。</li>
		<li>让孔在室温下涂覆至少15分钟。</li>
		<li>使用真空吸气器，小心地从孔中去除任何剩余的明胶（倾斜板以确保去除所有液体而不会刮掉明胶层）。</li>
		<li>向每个孔中加入 0.5 mL NBiL 培养基（步骤 1.3），并将板留在组织培养箱中预热。</li>
	</ol></li>
	<li>传代 mPSC。<ol><li>将 30 mL 洗涤介质（步骤 1.5）等分装到 50 mL 锥形管中。</li>
		<li>从mESC的组织培养皿中吸出旧培养基。 注意：允许使用多种抽吸技术。无论选择哪种技术，都要确保细胞不会长时间保持干燥（最多约 30 秒）。</li>
		<li>加入所需量的市售细胞分离培养基（10 cm 培养皿为 1.5 mL，相应缩放，参见 材料表）。</li>
		<li>等待3-5分钟，然后用P1000移液器上下移液15-20次，以获得单细胞悬浮液。在显微镜下观察细胞，以确保单细胞悬浮液。</li>
		<li>将分离培养基中的细胞转移到含有洗涤培养基的 50 mL 试管中。</li>
		<li>在室温下以300× g 离心5分钟。吸出洗涤介质，确保试管中没有残留液体。</li>
		<li>将沉淀重悬于少量洗涤介质（~300μL）中。使用血细胞计数器计数细胞悬液以获得细胞密度。</li>
	</ol></li>
</ol>4. mPSCs的反向转染
<ol><li>根据步骤2制备多转染试剂。</li>
	<li>根据步骤 3 制备细胞培养容器和传代 mPSC。</li>
	<li>将 200 μL NBiL 培养基等分到 1.5 mL 试管中，每次处理一个。</li>
	<li>向 DNA：OptiMEM 混合物中加入 26 μL OptiMEM：转染试剂混合物。通过移液约10次快速而剧烈地混合试剂。让混合物在室温下孵育5分钟。</li>
	<li>根据细胞密度，将所需体积的细胞从 300 μL 细胞悬液转移到 200 μL NBiL 管中，以获得每管 25,000 个细胞。</li>
	<li>将 Lipofectamine 2000（L2K，转染试剂）和 DNA 混合物孵育 5 分钟后，将其加入 1.5 mL 细胞管中，并通过移液充分混合。让细胞在室温下与试剂一起孵育5分钟。</li>
	<li>在室温下以300× g 离心5分钟。移出液体，剩下约 50 μL。</li>
	<li>用 100 μL NBiL 培养基重悬细胞沉淀。将细胞转移到培养皿中并将培养箱中放置。24 小时后使用新鲜的 NBiL 培养基更换培养基。</li>
</ol>5. mPSCs的正向转染
<ol><li>转染前12-18小时，根据步骤3制备细胞培养容器和传代mPSC。</li>
	<li>根据细胞密度，将所需体积的细胞从 300 μL 细胞混合物中转移到每孔接种 25,000 个细胞。将培养板放入培养箱中。</li>
	<li>转染前 1 小时，从所有孔中抽吸培养基并用 0.5 mL NBiL 培养基代替。将培养板放入培养箱中。</li>
	<li>转染时，按照步骤2制备mPSC多转染试剂。</li>
	<li>向 DNA：OptiMEM 混合物中加入 26 μL OptiMEM：转染试剂混合物。通过移液约10次快速而剧烈地混合试剂。让混合混合物在室温下孵育 5 分钟。</li>
	<li>将混合混合物滴加到孔中。将培养板放入培养箱中。24 小时后更换介质。</li>
</ol>6. 流式细胞术
<ol><li>转染后48小时从转染的24孔板中吸出培养基。</li>
	<li>向每个孔中加入200μL胰蛋白酶，涡旋，并在37°C下孵育30秒。 敲击板的侧面并加入 200 μL 冰冷的 FACS 缓冲液（PBS 中的 2% FBS）。</li>
	<li>从 A1 开始，打开并标记 V 底 96 孔板。在转染板上混合每个孔的内容物以去除细胞，并用 P200 移液管制备单细胞溶液。将 200 μL 从每个孔转移到 96 孔板上的相应孔中。</li>
	<li>将 15 mL FACS 缓冲液加入 50 mL 试剂储槽中。在室温下以300× g 离心板5分钟。通过将 96 孔板倒入水槽中以快速平稳的运动弃去上清液。</li>
	<li>使用多通道移液器将沉淀重悬于试剂储液槽的 150 μL FACS 缓冲液中，重悬于 96 孔板上。重复步骤 6.4-6.5 两次。将 96 孔板放入避光的装满冰的盒子中。</li>
	<li>通过流式细胞仪运行样品以获得荧光报告基因的群体分布。<ol><li>确保细胞仪在激光和滤光片组合方面适合进行实验（例如，如果无法激发或检测远红外光谱，则不要使用红外报告基因）。</li>
		<li>包括用于标准化磁珠的其他样品，以便在分析过程中对数据进行 MEFL 转换。确保每个样品收集足够的细胞进行适当的统计分析9.</li>
	</ol></li>
	<li>从原始细胞仪文件中转换荧光单位，并使用几种方法中的任何一种分析数据，例如基于Python或MATLAB的管道或市售软件9,13。</li>
</ol>

mPSC 正向和反向转染方法的比较分析

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作者报告没有利益冲突。

转染方案被广泛采用的一个关键原因是其可重复性和可及性;然而，这些协议确实需要跨实验环境进行优化。上述未提及的是首次尝试转染新细胞系时所需的标准测试。首先，转染试剂的选择是关键，因为市售试剂不是万能的，而且不同细胞类型的NA递送活力效率会有所不同。此外，要找到NA和转染试剂的理想量及其最佳比例，还需要进行测试。通常遵循转染试剂供应商推荐的优化步骤就足以达到NA...

将 DNA 和 RNA 递送至哺乳动物细胞是生物医学研究的核心支柱1.将外源核酸 （NA） 引入哺乳动物细胞的常用方法是瞬时转染 2,3。该技术依赖于将 NA 与能够将其递送至受体细胞的市售转染试剂混合。通常，NA 通过正向转染递送，其中粘附在二维表面的细胞接受转染复合物。虽然对最常见的已建立细胞系进行正向转染是稳健的，并且实验方案已广泛发表，但更多具有非单层形态的利基细胞类型不容易转染，从而限制了可递送的 NA 量和接收它的细胞数量。
多能干细胞 （PSC） 是理解发育的有吸引力的模型，也是再生医学的工具，因为它们能够无限分裂并产生任何身体细胞类型。对于小鼠 PSC （mPSC），具有 2 种抑制剂和 LIF （2i/LIF） 的常规体外培养条件保持圆顶状集落形态，直接限制暴露于正向转染的细胞数量 4,5,6。为了解决这个问题，可以进行反向转染：将细胞加入含有培养基和转染试剂的培养皿中，而不是将转染试剂添加到贴壁细胞中 7。虽然这增加了暴露于试剂的细胞数量，但也需要同时传代和转染细胞。
除了简单的单NA转染之外，研究人员通常还致力于在体外将几种NA构建体递送至细胞群中。这通常通过共转染实现，其中 NA 以给定比例（1：1、9：1 等）混合，然后与所选转染试剂结合 8。这会产生 NA 和试剂的混合物，该混合物保留了 NA 彼此之间的原始比例 - 虽然处理中的细胞可能接受不同量的这种混合物，但它们都接受相同的比例9。虽然当知道所需的零件比例时，这是有利的，但提前确定该比例可能是劳动密集型的，每个比率都构成不同的条件。一种替代方法是进行“多转染”，即将单个 NA 与转染试剂彼此独立地混合9。通过组合含有单个 NA 的转染复合物（而不是在创建复合物之前组合 NA），研究人员可以在单次转染实验中探索各种 NA 化学计量学9。这在预计多个 NA 的产物会相互作用的情况下特别有价值，例如与诱导型转录系统或具有内置反馈的系统 1,10,11。然而，要有效地做到这一点，需要高转染效率。事实上，随着独特转染 NA 数量的增加，给定细胞接收所有所需 NA 的概率呈指数下降 9,12。
以下报告描述了使用基于阳离子脂质的转染试剂的 mPSC 反向转染方案，其中细胞暴露于试剂-NA 混合物中最多 5 分钟，以最大限度地提高活力并最大限度地减少典型培养条件之外的时间。将该方案与这些细胞的标准正向转染进行比较，显示出更高的转染效率和存活转染细胞总数的增加。通过将这种反向转染与涉及简单荧光报告基因的三质粒多转染相结合，证明了以高转染效率筛选 NA 比率的扩展潜力。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase&#160;</td>
			<td>MilliporeSigma</td>
			<td>SCR005</td>
			<td></td>
		</tr>
		<tr>
			<td>Apotransferrin&#160;</td>
			<td>MilliporeSigma</td>
			<td>T1147-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA fraction V (7.5%)</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021&#160;</td>
			<td>MilliporeSigma</td>
			<td>SML1046-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>MilliporeSigma</td>
			<td>D6421-24X500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry standardization beads</td>
			<td>Spherotech</td>
			<td>URCP-38-2K</td>
			<td></td>
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		<tr>
			<td>Gelatin&#160;</td>
			<td>MilliporeSigma</td>
			<td>G1890</td>
			<td></td>
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			<td>GlutaMAX supplement&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>35050061</td>
			<td></td>
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			<td>Insulin&#160;</td>
			<td>Gibco</td>
			<td>12585-0014</td>
			<td></td>
		</tr>
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			<td>Lipofectamine 2000&#160;</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM&#160;</td>
			<td>Invitrogen</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>PD0325901&#160;</td>
			<td>MilliporeSigma</td>
			<td>PZ0162-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>MilliporeSigma</td>
			<td>P8783</td>
			<td>Chemical hazard - consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>MilliporeSigma</td>
			<td>P6780</td>
			<td>Chemical hazard - consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood</td>
		</tr>
		<tr>
			<td>Recombinant mLIF&#160;</td>
			<td>BioTechne</td>
			<td>8878-LF-500/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite&#160;</td>
			<td>MilliporeSigma</td>
			<td>S5261-25G</td>
			<td>Chemical hazard - consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized mouse embryonic stem cell based reverse poly-transfection technique for rapid exploration of nucleic acid ratios

由于其相对简单和易用性，用核酸瞬时转染哺乳动物细胞系已成为生物医学研究的支柱。虽然大多数广泛使用的细胞系在贴壁二维培养物中具有可靠的转染方案，但这些方案通常不能很好地转化为研究较少的细胞系或具有非典型、难以转染形态的细胞系。该方法使用在2i/LIF培养基中生长的小鼠多能干细胞（一种广泛用于再生医学的培养模型），概述了一种优化的快速逆转录转染方案，能够实现更高的转染效率。利用该方案，进行三质粒多转染，利用质粒递送中高于正常效率的效率来研究范围更广的质粒化学计量。这种反向多转染方案允许采用一锅实验方法，使用户能够在单个孔中优化质粒比率，而不是在多个共转染中优化质粒比率。通过促进快速探索DNA化学计量对递送遗传回路整体功能的影响，该协议最大限度地减少了胚胎干细胞转染的时间和成本。

Delivery of DNA and RNA into mammalian cells serves as a core pillar of biomedical research1. A common method for introducing exogenous nucleic acids (NA) into mammalian cells is through transient transfection2,3. This technique relies on mixing NA with commercially available transfection reagents capable of delivering them into the recipient cells. Typically, NA is delivered via forward transfection, where cells adhering to a two-dimensional surface receive the transfection complex. While forward transfection for the most common established cell lines is robust and protocols are well-published, more niche cell types with non-monolayer morphologies do not transfect easily, limiting the amount of NA that can be delivered and the number of cells that receive it.
Pluripotent stem cells (PSCs) serve as an attractive model for understanding development and as a tool for regenerative medicine, given their ability to divide indefinitely and produce any bodily cell type. For mouse PSCs (mPSCs), routine in vitro culture conditions with 2 inhibitors and LIF&#160;(2i/LIF) maintain a dome-like colony morphology, directly limiting the number of cells exposed to a forward transfection4,5,6. To address this, a reverse transfection can be performed: cells are added to a dish containing media and transfection reagent, rather than adding transfection reagent to adherent cells7. While this increases the number of cells exposed to the reagent, it also requires the cells to be passaged and transfected concurrently.
Moving beyond simple single-NA transfections, researchers often aim to deliver several NA constructs into a population of cells in vitro. This is typically achieved through a co-transfection, where the NAs are mixed at a given ratio (1:1, 9:1, etc.) and are then combined with the chosen transfection reagent8. This yields a mix of NAs and reagent that preserves the original ratio of NAs to one another - while cells in the treatment may receive different amounts of this mix, they all receive the same ratio9. While this is advantageous when the desired ratio of parts is known, determining this ratio ahead of time can be labor-intensive, with each ratio constituting a different condition. One alternative is to perform a &#34;poly-transfection,&#34; where individual NAs are mixed with the transfection reagent independently from one another9. By combining transfection complexes containing individual NAs (rather than combining NAs before creating the complexes), researchers can explore a wide array of NA stoichiometries in a single transfection experiment9. This is particularly valuable in cases where the products of several NAs are expected to interact with one another, such as with inducible transcription systems or systems with feedback built in1,10,11. However, to do so effectively, a high transfection efficiency is needed. Indeed, as the number of unique transfected NAs increases, the probability of a given cell receiving all of the desired NAs decreases exponentially9, 12.
The following report describes a reverse transfection protocol for mPSCs using a cationic lipid-based transfection reagent, in which cells are exposed to the reagent-NA mix for a maximum of 5 min to maximize viability and minimize the time outside of typical culture conditions. Comparing this protocol to the standard forward transfection of these cells demonstrates a higher transfection efficiency and an increase in the total number of surviving transfected cells. By combining this reverse transfection with a three-plasmid poly-transfection involving simple fluorescent reporters, an expanded potential to screen NA ratios with high transfection efficiency is demonstrated.

作者聚焦：用于干细胞命运操纵和细胞疗法开发的合成基因装置的进展

一种基于小鼠胚胎干细胞的优化反向多转染技术，用于快速探索核酸比率

Our work generally focuses on using synthetic genetic devices to manipulate stem cell fate and behavior. By controlling cellular states, we aim to advance the development of next generation cellular therapies. Recently, a deeper understanding of the relationship between the dosage of a fate determining gene and the cell's lineage has been established.This was facilitated by the development of genetic controllers that are able to titrate a given gene precisely. Classic molecular biology techniques such as transfection or lentiviral transduction for simple gene over expression will always be a mainstay. However, our field has recently adopted synthetic devices that incorporate linear and non-linear feedback in order to obtain more complex outputs from delivered DNA.Gene dosage has a key role in determining the outcome of gene expression. When delivering multiple exogenous genes, a key experimental factor is how much of each gene will give the optimal or desired outcome. Our protocol allows researchers to rapidly scan across dosages in a single treatment, looking for the desired ratio of DNA devices.

正向和反向转染都依赖于细胞膜与传入的转染试剂-DNA 复合物之间的相互作用，从而允许将 NA 递送至受体细胞。这些技术的不同之处在于细胞递送时的状态 - 在传统的正向转染中，DNA通常递送至贴壁细胞单层，而反向转染则依赖于试剂-DNA复合物在单细胞悬浮液中与细胞相遇。在细胞不以均匀、扁平的单层形式生长，而是采用更圆顶或菌落状形态的情况下，这种差异尤其重要，就像 mPSC 一样。可以...

Watch this Scientific Journal Video about An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios at JoVE.com

本方案描述了一种在用2i和LIF培养基培养过程中对小鼠胚胎干细胞进行反向多转染的方法。与传统的正向转染方案相比，该方法具有更高的活力和效率，同时还能够实现质粒比率的一锅优化。

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical ...

我们的工作通常集中在使用合成遗传设备来操纵干细胞的命运和行为。通过控制细胞状态，我们的目标是推进下一代细胞疗法的发展。最近，对决定命运的基因的剂量与细胞谱系之间的关系有了更深入的了解。这得益于能够精确滴定给定基因的遗传控制器的开发。经典的分子生物学技术，如转染或慢病毒转导，用于简单的基因过表达，将始终是中流砥柱。然而，我们的领域最近采用了结合线性和非线性反馈的合成设备，以便从递送的DNA中获得更复杂的输出。基因剂量在决定基因表达的结果方面起着关键作用。当递送多个外源基因时，一个关键的实验因素是每个基因中有多少将给出最佳或期望的结果。我们的方案允许研究人员在单次治疗中快速扫描不同剂量，寻找所需的DNA设备比例。

an-optimized-mouse-embryonic-stem-cell-based-reverse-poly

Research

JoVE Journal

Developmental Biology

An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios

692 次观看.  University of British Columbia. 我们的工作通常集中在使用合成遗传设备来操纵干细胞的命运和行为。通过控制细胞状态，我们的目标是推进下一代细胞疗法的发展。最近，对决定命运的基因的剂量与细胞谱系之间的关系有了更深入的了解。这得益于能够精确滴定给定基因的遗传控制器的开发。经典的分子生物学技术，如转染或慢病毒转导，用于简单的基因过表达，将始终是中流砥柱。然而，我们的?...

视频: 作者聚焦：用于干细胞命运操纵和细胞疗法开发的合成基因装置的进展

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical research. While most widely used cell lines have robust protocols for transfection in adherent two-dimensional culture, these protocols often do not translate well to less-studied lines or those with atypical, hard-to-transfect morphologies. Using mouse pluripotent stem cells grown in 2i/LIF media, a widely used culture model for regenerative medicine, this method outlines an optimized, rapid reverse transfection protocol capable of achieving higher transfection efficiency. Leveraging this protocol, a three-plasmid poly-transfection is performed, taking advantage of the higher-than-normal efficiency in plasmid delivery to study an expanded range of plasmid stoichiometry. This reverse poly-transfection protocol allows for a one-pot experimental method, enabling users to optimize plasmid ratios in a single well, rather than across several co-transfections. By facilitating the rapid exploration of the effect of DNA stoichiometry on the overall function of delivered genetic circuits, this protocol minimizes the time and cost of embryonic stem cell transfection.

The present protocol describes a method for reverse poly-transfection of mouse embryonic stem cells during culture with 2i and LIF media. This method yields higher viability and efficiency than traditional forward transfection protocols, while also enabling one-pot optimization of plasmid ratios.

科研

发育生物学

Preparation of Mouse Pluripotent Stem Cells &#40;mPSCs&#41; for Transfection

To prepare a gelatin coated 24 well plate add 200 microliters of the 0.2%gelatin solution to each well. Gently shake the plate to evenly spread the solution and allow the wells to coat at room temperature. After 15 minutes, using a vacuum aspirator carefully remove any leftover gelatin from the wells.Add 0.5 milliliters of freshly prepared NBIL media to each well. And place the plate in a tissue culture incubator to prewarm. Aliquot 30 milliliters of wash media into a 50 milliliter conical tube.Aspirate the media from the tissue culture dish of mESCs and add required amount of cell detachment medium. After three to five minutes, pipette up and down 15 to 20 times using a P1, 000 pipette. Observe the cells under a microscope.To ensure a single cell suspension transfer the cell suspension into a 50 milliliter tube containing wash media. Centrifuge the suspension at 300G for five minutes at room temperature and aspirate the wash media. Resuspend the pellet in approximately 300 microliters of wash media.

用于转染的小鼠多能干细胞（mPSCs）的制备

Reverse and Forward Transfection of Mouse Pluripotent Stem Cells &#40;mPSCs&#41;

To begin, aliquot 200 microliters of NBIL media into 1.5 milliliter tubes. Add 26 microliters of the optimum transfection reagent mixture to the DNA optimum mixture. Pipe at the reagents vigorously approximately 10 times.Transfer the required volume of mPSC suspensions to the 200 microliters of NBIL tubes. Then add lipofectamine 2000 and DNA mixture to the tubes and mix well by pipetting. After five minutes, centrifuge the mixture at 300 G for five minutes and remove the supernatant, leaving approximately 50 microliters in the tube.Re suspend the pellet in 100 microliters of NBIL media. Transfer the cell suspension to a gelatin-coated 24-well plate and place plate in the incubator. Transfer the required volume of mPSC suspension to seed 25, 000 cells per well of a gelatin coated 24-well plate and place the plate in the incubator.One hour prior to transfection. Replace the media from the wells with 0.5 milliliters of NBIL media. After incubation, dropwise, add lipofectamine 2000 and DNA mixture to the wells.Place the plate in the incubator for 48 hours.

小鼠多能干细胞（mPSCs）的逆转录和正向转染

Flow Cytometry Analysis of Transfected Mouse Pluripotent Stem Cells &#40;mPSCs&#41;

After transfection of mPSCs, aspirate the media from the six-well plate. Then add 200 microliters of trypsin into each well. Swirl and incubate for 30 seconds at 37 degrees Celsius.Tap the side of the plate and add 200 microliters of ice cold FACS buffer. Using a P200 pipette, mix the contents of each well to dislodge cells and make single cell solutions. Transfer 200 microliters from each well to the appropriate well on the 96-well plate Centrifuge the plate at 300 G for five minutes.After centrifugation, resuspend the pellets in 150 microliters of FACS buffer before running the sample on a flow cytometer. Compared to reverse transfections, forward transections showed a significantly lower proportion of cells positive for the marker. Interestingly, forward Transfect did not deliver a significantly lower amount of DNA on average to the population of cells.In reverse transections, incubation time significantly influenced the percentage of reporter positive cells. However, the average reporter expression in positive cells was not affected by incubation time. For both poly and co-transfection, a forward transfection significantly decreased the number of cells present at the chosen endpoint compared to reverse transfections.In the case of reverse poly-transections, higher transfection efficiency and a broader dynamic range of well-represented DNA ratios were observed.