我们感谢Gi Bum Kim的免疫荧光染色和图像。我们还要感谢 Emily Hatanaka、Naomi Kobritz 和 Paul Linesch 对协议的有用建议。

该协议中的所有程序均已获得 Cedars Sinai 医疗中心机构动物护理和使用委员会 （IACUC） 的批准。纯合 子mTmG 小鼠与C57BL / 6J小鼠一起繁殖，以获得混合性别，杂合 子mTmG 小鼠的窝，用于以下方案。这些动物是从商业来源获得的（见 材料表）。在出生后第 0 天和第 3 天 （P0-P3） 之间对小鼠幼崽进行电穿孔。
1. 手术设置
<ol><li>用化学消毒剂（例如 Vimoba）喷洒手术台并擦拭，然后用 70% 乙醇擦拭，然后再次擦拭。</li>
	<li>将以下器械/材料放在手术台上：拉动的玻璃毛细管移液器（有关如何拉动移液器的信息见参考文献10 ）、小剪刀、小型生物危害锐器废物容器、微升移液器和移液器吸头、2 块切开的方形石蜡膜、电极凝胶、电极、显微注射器支架和 DNA 质粒混合物（参见 材料表 和 补充文件 1）。</li>
	<li>指示时将以下附件放置在手术台上或地板上的设备上：显微注射器控制面板、支架和连接到机器的插头、用于将显微注射器支架固定在侧面的支架（助焊器）、显微注射器脚踏板（在地板上）、连接到机器的电极和电极脚踏板（在地板上）（参见 材料表）。 注意： 电极脚踏板放置在显微注射器脚踏板的右侧，以提醒使用顺序。</li>
	<li>打开显微注射器和电极控制面板。<ol><li>检查显微注射器上的设置是否适合实验：压力必须在 220-450 hPa 之间，具体取决于质粒混合物的吸收方式。</li>
		<li>检查电极面板上的设置是否适合实验。 注意：对于当前的实验（以及我们的大多数脑肿瘤模型），使用五个120 V脉冲（50 ms;间隔950 ms）。</li>
	</ol></li>
</ol>2. 术前准备
<ol><li>在显微注射器中制备质粒混合物。<ol><li>取回一个拉动的玻璃毛细管移液器并将其插入显微注射器支架中。确保尖端已拧紧以固定到位。</li>
		<li>一只手握住显微注射器支架，另一只手握住小剪刀;将移液器的尖端放在一个小的锐器生物危害容器上。在剪刀合上的情况下，轻轻按下加长的玻璃毛细管移液管。在看到弯曲的地方切割，距离开口约 2 毫米。如果切割看起来成功，请小心地放在一边。</li>
		<li>将市售的质粒混合管平放在手术台上并打开。将移液器吸头盒或其他物品放在试管底部后面，以便在吸取质粒混合物时保持稳定。将连接到显微注射器管路的玻璃移液管平放在桌子上，然后轻轻插入质粒混合物的管中，确保尖端在靠近管底部的混合物中。 注意：由于自然重力抽吸，质粒混合物抽吸将在玻璃移液器中开始，然后主动吸入。</li>
		<li>将玻璃移液管的尖端放入质粒混合物中，使用显微注射器面板增加吸入量。从 0 开始，转动右旋钮并朝 -60 的负方向拨动 - 这会将质粒混合物带入玻璃移液器。仅引入 ~1 英寸的质粒混合物。将旋钮转回 0，然后从混合物中取出移液器吸头。 注意：不要在负方向上从混合物中取出移液器吸头，因为这会将混合物射入显微注射器并可能射入管道。</li>
		<li>通过将混合物注射到一条石蜡薄膜上来测试一次注射中的混合物量。使用脚踏板并按一次将混合物注入石蜡膜上。使用设置为1μL的移液器在石蜡膜上吸取混合物，看看它是否正好是1μL。 注意：如果略小于 1 μL，则可以增加显微注射器的压力。如果压力接近 450 hPa 但仍低于 1 μL，则玻璃移液器开口太小，需要修剪更多。建议在修剪前将质粒混合物放回试管中。尽管如此，剪刀上仍然会有残留的混合物，因此必须用 DNase 彻底清洁剪刀以去除任何残留的混合物。如果进样量超过1 μL，则需要使用新的拉动玻璃移液器并进行修剪。当测试量正好为 1 μL 时，玻璃移液器吸头的尺寸合适。</li>
		<li>重复步骤2.1.4，为该过程吸入额外的质粒混合物，或将约2-3英寸吸入拉动的玻璃移液器中。确保混合物不会太深入显微注射器，在那里看不到末端。 注意：吸入质粒混合物时，避免气泡至关重要。如果存在气泡，则必须通过从拉出的玻璃移液管中取出质粒混合物并重新开始来重做此步骤。当注射到幼犬的心室时，存在的气泡将对以下步骤有害。</li>
		<li>在拉动的玻璃移液器中准备好质粒混合物后，将准备好的移液器放在助焊器支架的一侧并移开，以免意外接触/碰撞它。</li>
	</ol></li>
	<li>准备动物进行实验。<ol><li>准备一个冰桶来麻醉幼崽。向冰桶中加水使其融化并帮助冷却麻醉支架（例如，移液器盒顶部）。</li>
		<li>将盒子顶部放在融化的冰上冷却。</li>
		<li>小心地将幼崽从笼子中取出，并将它们放入冷却的箱顶中，箱顶仍留在冰上。将另一个顶部松散地放在底部以帮助冷却过程。</li>
		<li>2-3分钟后，检查幼崽。将幼崽彼此分开，同时保持俯卧位。 注意：幼崽会蠕动，但随着冷麻醉的开始，这会减慢速度。箱顶可以省略，以便于查看幼崽。</li>
		<li>要检查麻醉是否正确，请捏住幼犬的尾巴并寻找反应。如果有反应，重复步骤2.2.4。如果没有尖叫声或很少移动，幼犬就可以进行手术了。 注意：小鼠不应长时间放在冰上，因为这会影响它们在手术后的恢复能力。一次只能将麻醉下既可以注射又可以电穿孔的幼崽数量放在冰上;通过经验可以完成更多的工作。</li>
	</ol></li>
</ol>3. 将DNA质粒混合物显微注射到脑室
<ol><li>将一只小狗放在腹侧位置的桌子上。</li>
	<li>稍微向后拉头部后部，以观察颅骨缝合线穿过皮肤。在头骨上找到lambda。心室/注射部位将位于 lambda 和眼睛之间。 注意：Lambda 是矢状缝合线和羊角缝合线相交的地方。参见参考文献11 以鉴定小鼠头骨上的 lambda。</li>
	<li>用玻璃移液器吸头以垂直角度刺穿皮肤（移液器笔直朝向天花板）。按压颅骨时观察轻微的凹陷和初始阻力。一旦刺穿并且玻璃移液器吸头戳穿凹陷，即达到适当的注射水平。</li>
	<li>将玻璃移液器吸头固定到位，并按下显微注射器脚踏板一次，以注射1μL质粒混合物。 注意：有两种方法可以清楚地看到注射成功。首先，让另一名实验室成员观察质粒混合物在注射时在玻璃移液器中下降。此外，如果在质粒混合物中使用快速绿色或其他染料，一旦注射，染料就会在皮肤下的心室中明显扩散，并且当拿到手术灯时很容易看到。</li>
</ol>4. 电穿孔
<ol><li>将一块石蜡膜（一个正方形）放在桌子上，然后挤出薄膜顶部的电极凝胶。在每个电极片上刷大量，将电极片盖在电极凝胶中。任何滴落的多余凝胶都可以在纸巾上擦掉。 注意：在第一次 EP 之后，不要让每个桨的凝胶接触另一侧。这将转移电荷，当应用于幼犬的头部时，可能会听到嘶嘶声。如果来自一个电极的幼犬头上的凝胶与来自另一个电极的凝胶接触，也会发生这种情况。 注意：在电穿孔过程中，必须注意使手指远离桨叶。</li>
	<li>用一只手（通常是非惯用手）握住幼犬的身体，将手指保持在头部后面。 注意： 如果是右撇子，最容易用左手握住幼犬，用右手握住电极。如果是左撇子，则反其道而行之。</li>
	<li>将电极靠近幼犬的头部，以确保它们处于正确的位置，正极位于头部外侧，即心室被瞄准的地方（例如，如果瞄准左心室，则将正极放在幼犬的左侧，负极放在幼犬的右侧）。定位电极时，轻轻滑动手指（通常是拇指）在头部后方的身体/颈部背侧，以帮助将头部抬高到位。</li>
	<li>通过捏住尾巴来检查麻醉深度，并且仅在幼犬处于适当的麻醉平面时才进行电穿孔。轻轻挤压鼠标头部喙部的电极桨，将它们放在眼睛上。踩一次电极脚踏板开始电穿孔。该协议使用五个 120 V 脉冲（50 ms，间隔 950 ms）;每个脉冲都会被听到。<ol><li>随着每个脉冲的产生，沿逆时针方向稍微旋转电极桨，使正电极桨向头顶移动。 注意：通过适当的电穿孔，人们会感觉到/看到小鼠幼崽的身体随着每个脉冲而抽搐。</li>
	</ol></li>
	<li>在最后一次脉冲之后，取下桨叶并放在一边。将幼犬移到干净的纸巾上，让另一名实验室成员清除幼犬头上的凝胶，然后放在纸巾“船”上的加热灯下。 注意： 确保加热 lamp 不要离幼崽太近。确保有另一位实验室成员帮助完成此步骤，并持续关注幼崽。在加热灯下将幼崽握在手中以增加幼崽的热量通常会有所帮助。轻轻按摩幼犬的腹部也有助于恢复。</li>
	<li>一旦幼崽的身体呈健康的粉红色，并且对轻微的尾巴捏捏有吱吱声，就将它们放回笼子。 注意： 在将它们放回笼子之前，取少量家庭笼子垫料，并用它轻轻刷拭幼崽以获得笼子的气味。将幼崽放回笼子里，放在垫料下，然后返回饲养室。</li>
</ol>5. 术后步骤
<ol><li>将未使用的质粒混合物从显微注射器玻璃移液器放回试管中。如果还剩下足够的混合物，则可以在以后的程序中使用。储存在-20°C。</li>
	<li>用纸巾擦去电极桨上的凝胶。</li>
	<li>将所有设备部件放回原处。 注意：如果在吸收质粒混合物 后 使用剪刀剪断玻璃移液器尖端，请将剪刀放在外面用DNase溶液彻底清洁。</li>
	<li>用化学消毒剂喷洒在桌子上，然后擦拭干净，然后喷洒 70% 乙醇，然后再次擦拭。</li>
</ol>

通过基于电穿孔的质粒 DNA 递送进行体内脑肿瘤建模

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作者没有什么可透露的。

基于电穿孔的质粒DNA递送允许在体内使用分子生物学，类似于基因工程小鼠模型中使用的分子生物学，但具有病毒转导的速度、定位和效率8,13,14。然而，后者带来了安全问题和免疫反应。我们在使用质粒 DNA 的 EP 递送的建模系统中表明，由于最初将玻璃毛细管移液管插入脑室8，因此发生了最小的免疫?...

小鼠脑肿瘤模型对于理解脑肿瘤形成和治疗的机制至关重要。目前的模型通常包括基于有限数量的驱动突变或患者来源的异种移植模型，使用阻碍适当免疫治疗研究的免疫缺陷小鼠对常用肿瘤细胞系进行快速产生的皮下或原位移植 1,2,3,4。此外，这些临床前结果可能导致假阳性，因为此类模型可以表现出对治疗反应的戏剧性，通常是治愈效果，但这并不能转化为临床 2,5,6,7。能够快速生成更能反映患者突变特征的基因工程临床前小鼠模型对于提高临床前结果的有效性至关重要。
基于电穿孔 （EP） 的 DNA 质粒递送可诱导功能丧失 （LOF） 和功能获得 （GOF） 突变，从而可以生成此类模型。我们开发了一种更精确地表示 GOF 驱动突变的方法，称为双重组酶介导的盒交换或 MADR8 的镶嵌分析。该方法允许在体细胞中以受控的、位点特异性的方式表达感兴趣的基因（或多个基因）8。结合其他分子工具，如成簇的规则间隔短回文重复序列（CRISPR），可以将不同的患者突变结合起来，开发小鼠脑肿瘤模型。该方法已用于不同的小儿脑肿瘤，包括胶质瘤和室管膜瘤8，以及成人脑肿瘤模型，例如胶质母细胞瘤 （GBM）。
虽然肿瘤建模的EP方法不像移植那样普遍，但以下证明了迄今为止该建模系统的易用性和高可重复性。mTmG小鼠用于插入MADR质粒DNA 8,9。该系统允许位于 Rosa26 位点的 loxP 和 Flp 重组酶靶标 （FRT） 位点进行重组，以便随后插入供体 DNA 质粒（即目的基因 GOF）8,9。以下方案证明了该方法在勤奋实践后的简单性，以及以自主，一致的方式开发小鼠脑肿瘤模型的能力。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1-2.5 &#181;L 1-channel pipette</td>
			<td>Eppendorf</td>
			<td>3123000012</td>
			<td></td>
		</tr>
		<tr>
			<td>2 &#181;L pipette tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-442</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;L pipette tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-432</td>
			<td></td>
		</tr>
		<tr>
			<td>2-20 &#181;L 1-channel pipette</td>
			<td>Eppendorf</td>
			<td>3123000098</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAZap PCR DNA Degradation Solutions</td>
			<td>Fisher Scientific</td>
			<td>AM9890</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM 830 Square Porator Electroporator</td>
			<td>BTX</td>
			<td>45-0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode Gel</td>
			<td>Parker Labs</td>
			<td>PLI152CSZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green Dye</td>
			<td>Sigma-Aldrich</td>
			<td>F7258-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Helping Hands Soldering Aid</td>
			<td>Pro&#39;sKit</td>
			<td>900-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors, 4.5&#34; Straight Sharp</td>
			<td>Roboz</td>
			<td>RS-5916</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Strain: C57BL/6J</td>
			<td>The Jackson Laboratory</td>
			<td>JAX: 000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Strain: Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J</td>
			<td>The Jackson Laboratory</td>
			<td>&#160;JAX: 007676</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Grainger</td>
			<td>16Y894</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid: pCag-FlpO-2A-Cre EV&#160;</td>
			<td>Addgene</td>
			<td>129419</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Tweezertrode, 7 mm Diameter</td>
			<td>BTX</td>
			<td>45-0488</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharps container, 1-quart</td>
			<td>Uline</td>
			<td>S-15307</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Glass Capillaries, 4 in, 1 mm OD, 0.58 mm ID</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td>Capillary pipettes need to be pulled - see reference 10 for details.&#160;</td>
		</tr>
		<tr>
			<td>Vertical Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-30</td>
			<td>Heat settings: Heat #1 at 880, Heat #2 at 680; pull at 800. See reference 10 for more details on pulling.&#160;</td>
		</tr>
		<tr>
			<td>Vimoba Tablet Solution</td>
			<td>Quip Laboratories</td>
			<td>VIMTAB</td>
			<td></td>
		</tr>
		<tr>
			<td>XenoWorks Digital Microinjector</td>
			<td>Sutter Instruments</td>
			<td>BRE</td>
			<td></td>
		</tr>
		<tr>
			<td>XenoWorks Micropipette Holder</td>
			<td>Sutter Instruments</td>
			<td>BR-MH</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling brain tumors in vivo using electroporation-based delivery of plasmid dna representing patient mutation signatures

肿瘤模型对于脑肿瘤的临床前测试至关重要，因为它可以探索新的、更有效的治疗方法。随着人们对免疫疗法的浓厚兴趣，拥有一致的、临床相关的、免疫功能正常的小鼠模型来检查大脑中的肿瘤和免疫细胞群及其对治疗的反应就显得尤为重要。虽然大多数临床前模型利用已建立的肿瘤细胞系的原位移植，但这里介绍的建模系统允许从 插入体内分裂神经前体细胞 （NPC） 的 DNA 构建体中逐步而有效地开发患者特异性肿瘤突变的“个性化”表示。DNA 构建体采用双重组酶介导的盒式交换 （MADR） 方法进行嵌合分析，可实现驱动突变的单拷贝体细胞诱变。使用出生至3天大的新生小鼠幼崽，通过利用侧脑室内衬的这些分裂细胞来靶向NPC。将 DNA 质粒（例如，MADR 衍生的转座子、CRISPR 定向的 sgRNA）显微注射到心室中，然后使用围绕头部喙部区域的桨进行电穿孔。在电刺激下，DNA被吸收到分裂细胞中，并有可能整合到基因组中。这种方法的使用已成功用于发展儿童和成人脑肿瘤，包括最常见的恶性脑肿瘤胶质母细胞瘤。本文讨论并演示了使用该技术开发脑肿瘤模型的不同步骤，包括麻醉年轻小鼠幼崽的过程，到质粒混合物的显微注射，然后进行电穿孔。有了这种自主的、免疫功能正常的小鼠模型，研究人员将有能力扩展临床前建模方法，以努力改进和检查有效的癌症治疗。

Murine brain tumor models are crucial for understanding the mechanisms of brain tumor formation and treatment. Current models typically include rapidly produced subcutaneous or orthotopic transplantations of commonly used tumor cell lines, based on a limited number of driver mutations or patient-derived xenograft models, using immunodeficient mice that hinder proper immunotherapy studies1,2,3,4. Additionally, these preclinical results can lead to false positives, in that such models can exhibit dramatic, oftentimes curative effects in response to therapy, but this does not translate to the clinic2,5,6,7. Having the ability to rapidly produce genetically engineered preclinical mouse models that are more reflective of patient mutation signatures is imperative for improving the validity of preclinical results.
Electroporation (EP)-based delivery of DNA plasmids to induce both loss of function (LOF) and gain of function (GOF) mutations allows for the generation of such models. We developed a method for an even more precise representation of GOF driver mutations called mosaic analysis with dual-recombinase-mediated cassette exchange, or MADR8. This method allows for the expression of a gene (or genes) of interest in a controlled, locus-specific manner in somatic cells8. In combination with other molecular tools, such as clustered regularly interspaced short palindromic repeats (CRISPR), different patient mutations can be combined to develop mouse brain tumor models. This method has been used for different pediatric brain tumors, including gliomas and ependymomas8, as well as adult brain tumor models, such as glioblastoma (GBM).
While the EP method of tumor modeling is not as common as a transplant, the following demonstrates heretofore the ease and high reproducibility of this modeling system. mTmG mice are used for the insertion of the MADR-plasmid DNA8,9. This system allows for the recombination of loxP&#160;and Flp recombinase target (FRT) sites located at the Rosa26 locus for subsequent insertion of the donor DNA plasmid (i.e., GOF gene of interest)8,9. The following protocol demonstrates the straightforwardness of this method after diligent practice, and the ability to develop mouse brain tumor models in an autochthonous, consistent manner.

作者聚焦：使用基于电穿孔的质粒 DNA 递送代表患者突变特征的体内脑肿瘤建模  

使用基于电穿孔的质粒DNA递送对 体内 脑肿瘤进行建模，以代表患者突变特征

The technique focuses on modeling patient mutation profiles of glioblastoma in the immunocompetent mouse model and how these different mutations are influencing the tumor microenvironment and response to treatment. Genomic and single cell sequencing identify that the genetic driver mutations of glioblastoma are associated with the aggressiveness of cancer and components of the tumor microenvironment such as the immune cell population. Glioblastoma mouse models in preclinical trials show great success with immunotherapy leading to curative results and no sign of tumor growth after treatment.However, these effects are not reflected in patients for outcome or survival. This protocol recapitulates patient tumor mutation profiles of glioblastoma in the immunocompetent mouse model, allowing gradual autochthonous tumor growth and better prediction of treatment efficacy, especially with immunotherapy. This modeling system integrates DNA plasmids into the genome through electroporation of immunocompetent mice.It avoids the use of viruses and transplanting thousands of tumor cells, both of which can have their own immune effects, confounding downstream results. Utilizing an immunocompetent mouse model that recapitulates patient mutation profiles allows for a more accurate assessment of treatment outcomes, preventing the laborious task of taking false positive results to the clinic.

上述方案已用于成功开发儿童和成人脑肿瘤小鼠模型，前者在Kim等人8中详细发表。通过适当的技术和对质粒设计的仔细规划，肿瘤EP开发的成功率通常为100%。当使用报告蛋白时，组织学是检查DNA质粒插入是否成功的最快和最简单的方法。该协议涉及如何开发具有 100% 外显率的 GBM 脑肿瘤模型的步骤，如组织学分析所证实的那样。MADR 供体质粒同时表达报告蛋白 （smTagBFP2-V5） 和 Sp...

Watch this Scientific Journal Video about Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures at JoVE.com

利用由常见患者突变驱动的免疫功能正常的本土肿瘤模型进行临床前测试对于免疫治疗测试至关重要。该协议描述了一种使用基于电穿孔的质粒DNA递送生成脑肿瘤小鼠模型的方法，这些质粒DNA代表常见的患者突变，从而提供准确，可重复和一致的小鼠模型。

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in ...

该技术的重点是在免疫功能正常的小鼠模型中模拟胶质母细胞瘤的患者突变谱，以及这些不同的突变如何影响肿瘤微环境和对治疗的反应。基因组和单细胞测序确定胶质母细胞瘤的遗传驱动突变与癌症和肿瘤微环境组成部分（如免疫细胞群）的侵袭性有关。临床前试验中的胶质母细胞瘤小鼠模型在免疫疗法方面取得了巨大成功，取得了治愈效果，治疗后没有肿瘤生长的迹象。然而，这些影响并未反映在患者的结局或生存率上。该方案概括了免疫活性小鼠模型中胶质母细胞瘤的患者肿瘤突变谱，允许逐渐的本土肿瘤生长并更好地预测治疗效果，尤其是免疫疗法。该建模系统通过对免疫功能正常的小鼠进行电穿孔将DNA质粒整合到基因组中。它避免了病毒的使用和移植数千个肿瘤细胞，这两者都可能产生自己的免疫作用，从而混淆下游结果。利用免疫活性小鼠模型来概括患者突变谱，可以更准确地评估治疗结果，避免将假阳性结果带到临床的艰巨任务。

modeling-brain-tumors-vivo-using-electroporation-based-delivery

Research

JoVE Journal

Cancer Research

Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures

1.0K 次观看.  Cedars-Sinai Medical Center. 该技术的重点是在免疫功能正常的小鼠模型中模拟胶质母细胞瘤的患者突变谱，以及这些不同的突变如何影响肿瘤微环境和对治疗的反应。基因组和单细胞测序确定胶质母细胞瘤的遗传驱动突变与癌症和肿瘤微环境组成部分（如免疫细胞群）的侵袭性有关。临床前试验中的胶质母细胞瘤小鼠模型在免疫疗法方面取得了巨大成功，取得了治愈效果，治疗后没有肿瘤生长的迹象?...

视频: 作者聚焦：使用基于电穿孔的质粒 DNA 递送代表患者突变特征的体内脑肿瘤建模  

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.

Utilizing an immunocompetent, autochthonous tumor model driven by common patient mutations for preclinical testing is critical for immunotherapeutic testing. This protocol describes a method to generate brain tumor mouse models using electroporation-based delivery of plasmid DNA that represent common patient mutations, thus providing an accurate, reproducible, and consistent mouse model.

科研

癌症研究

Microinjection of DNA Plasmid Mix into the Mouse Brain Ventricle

To begin, place the anesthetized mouse pup on the table in the ventral position. Pull the back of the pup's head slightly to visualize the cranial skull sutures through the skin. Find lambda on the skull.The ventricle or injection site will be visible midway between lambda and the eye. At a perpendicular angle, pierce the skin with a glass pipette tip, an initial resistance and slight concave indentation will be observed. Once pierced, hold the tip in place and press the micro injector foot pedal once to inject one microliter of the plasmid mix.

将DNA质粒混合物显微注射到小鼠脑室中

Electroporation-Based DNA Plasmids Delivery to Induce In Vivo Mutations in the Mouse

Begin electroporation-based DNA plasma delivery by placing a square piece of paraffin film on the table and squeezing the electrode gel on top of the film. Then apply a generous amount of the electrode gel on each paddle to cover the electrode paddles completely. Next, hold the pup micro-injected with the plasmid DNA mix in the non-dominant hand, keeping the fingers behind the head.To position the pup's head for electroporation, gently slide a finger on the next dorsal side, posterior to the head, and position the positive electrode outside the head where the ventricle is targeted. Place the electrode over the pup's eye and lightly squeeze the electrode paddles on the rostral part of the pup's head. Press the electrode foot pedal to start the electroporation.With each pulse, rotate the electrode paddles slightly, in a counterclockwise direction, so the positive paddle moves towards the top of the head. After the final pulse, remove the paddles and place them aside. Mice were moribund by five months post-electroporation, with full tumor growth detected through the reporter protein and histological analysis.