这份出版物的研究报告得到了国家卫生研究院的过敏和传染性疾病研究所 (NIAID) 的支持, 其奖项编号是 1R41OD021979 (对此) 和下一代 BioGreen 21 的研究补助金。计划, 大韩民国, 格兰特号PJ01107704。PJ01107703 (对 IK)。内容完全是作者的责任, 并不一定代表国家卫生研究院或 BioGreen 21 的官方观点。我们感谢 Dr. Nikolas Robl 编辑手稿。

本议定书所述程序由犹他州立大学动物保育和使用委员会 (IACUC) 批准 (IACUC 议定书: 2484)。本议定书使用的仓鼠是成人 (6-10 周龄) LVG 应变金黄叙利亚仓鼠。所有仓鼠都安置在犹他州立大学 Bioinnovation 中心的 vivarium。室温设置在23° c, 湿度设置为 40-50%, 光周期设置为 14 l:10 d (浅色: 深色)。只要有可能, 胚胎操作和 surugical 程序都应该用无菌技术进行。
1. sgRNA 和 Cas9/sgRNA 均一 (RNP) 的制备
<ol><li>合成 sgRNAs 后的体外转录程序在手册中描述的综合试剂盒 (材料表, 补充 1)。</li>
	<li>要组装均一, 混合2µg Cas9 与1µg sgRNA 和孵化混合在室温下为 10-15 分钟。对于 PN 注射液, 在浓度为 100 ng/µL Cas9 和 50 ng/µL sgRNA 与 10 mM TE 缓冲器 (核糖核酸自由,材料表) 的情况下, 制作一个工作解决方案。</li>
</ol>2. 输精管结扎术的准备
注: 输精管结扎术是在 6-8 周龄的雄性仓鼠身上进行的。手术应在第一次交配前10-14 天进行。不孕是证实的失败怀孕的交配与肥沃的女性。输精管男性通常可以使用一年前, 他们变得较少性活跃。
<ol><li>麻醉雄性经腹腔注射氯胺酮/嗪, 剂量为40毫克/千克 (氯胺酮) 和10毫克/千克 (嗪)。通过缺乏踏板反射 (脚趾捏) 来确认麻醉。</li>
	<li>用干纸巾把卧床的仓鼠放在背上。用70% 酒精和聚维酮碘之间交替的外科擦洗解决方案冲洗腹部三轮防腐处理。</li>
	<li>做一个垂直切口与外科剪刀开始 1.5 cm 头骨对从中间线延伸 1 cm 到每边的包皮 (图 1a)。切开皮肤、皮下组织和式, 进入腹膜间隙 (图 1b)。</li>
	<li>对阴囊的尾部施加轻柔的压力, 以迫使睾丸和脂肪组织出来。用镊子轻轻抓住输精管, 用第二组钳 (图 1c) 仔细地将血管从 vas 中分离出来。</li>
	<li>用钳夹住输精管形成一个循环。在火焰中加热另一个钳子的尖端, 直到它是红色的, 然后用它来除去输精管的回路。插入脂肪组织和睾丸回腹部。用相同的程序将对方的输精管切除。</li>
	<li>先缝合肌肉, 然后缝合皮肤。将动物放在笼中的暖垫上30分钟, 直到动物恢复。观察动物直到它恢复正常的活动。</li>
</ol>3. 供方/受援仓鼠准备时间表
<ol><li>监测和记录动情周期。 注: 雌性仓鼠有一个稳定的4天的动情周期, 在分娩后立即维持。雌性在发情的第一天可以通过不透明、黄色和粘稠的阴道分泌物 (图 2a) 来识别。4天发情的雌性可以通过透明的粘液 (图 2b) 进行识别。</li>
	<li>对于供体仓鼠的超数排卵, superovulate 在动情周期的第一天 (> 6 周), 在 9-上午12点之间, 通过 IP 注射妊娠母马血清促性腺激素 (PMSG; 在 PBS 中溶解为50毫升) (表 1)。 注: PMSG 用于诱导排卵, 但不适用于发情周期同步。</li>
	<li>80-84 h 在注射 PMSG (天 4, 6-下午8点) 以后, 安置女性在笼子与男性为交配。由于仓鼠交配不会导致交配插头, 确保成功的交配通过观看交配。</li>
	<li>在 4 天的发情与输精管男配性成熟女性准备 pseudopregnant 雌性。在表 2中显示了准备捐赠者和受赠女性的时间安排。 注意: 真正的怀孕女性也可以作为接受者,即, 女性与颜色不同的颜色与金色的颜色可育的雄性。从胚胎移植派生的幼崽可以根据涂层颜色来识别。使用真正的孕妇的一个潜在的优势是, 内生产的胚胎将确保成功的怀孕和帮助 PN 注射胚胎植入;使用真正的孕妇作为受体的潜在缺陷是, PN 注射胚胎需要与内生产的胚胎进行竞争。</li>
</ol>4. 受精卵分离
<ol><li>准备培养基 (HECM-9; 配方在补充2中描述), 如前面的麦基尔南和 Bavister12中所述。</li>
	<li>在 pn 注射前一天, 准备受精卵处理皿 (25 µL/滴) 和 pn 注射液滴 (100 µL) 在一个35毫米菜的盖子与 HECM-9。然后用矿物油覆盖中滴。在恒温箱中平衡培养基在以下条件下过夜: 37.5 ° c, 10% CO2, 5% O2, 85% N2和100% 湿度。</li>
	<li>在计划进行 PN 注射的当天, 用 HECM-9 准备胚胎冲洗皿 (1 碟/供体仓鼠), 并将所有的菜肴存放在孵化器中直到使用。</li>
	<li>安乐排仓鼠与 CO2。</li>
	<li>输卵管隔离<ol>
<li>将一只被安乐死的雌性仓鼠放在手术悬垂或纸巾上, 用70% 乙醇喷雾卧床腹部。</li>
		<li>在中线上牢牢握住皮肤和腹部肌肉层, 并进行切口。切开腹膜以暴露腹部器官。</li>
		<li>用细钳抓住其中一个子宫角, 从腹部缩回组织。将子宫与 mesometrium 和脂肪组织分开。在输卵管和卵巢之间进行切割, 并在输卵管和子宫的交界处 (包括子宫的一小部分) 附近进行第二次切割。将两个输卵管放在同一个冲洗盘中。</li>
	</ol>
</li>
	<li>冲洗和收集受精卵<ol>
<li>解剖显微镜下, 用 #5 的钳夹住子宫角的一侧, 将自制的针头 (图 3) 插入漏斗端的输卵管腔内, 用 300-400 µL 的 HECM-9 培养基冲洗输卵管。</li>
		<li>立即收集受精卵, 在处理盘中用 HECM-9 介质冲洗两次。在这个发展阶段, 所有的受精卵都应该从卵丘细胞中剥脱。</li>
		<li>将受精卵放入孵化器 (37.5 ° c, 10% CO2, 5% O2, 85% N2和100% 湿度) 后立即收集, 以尽量减少暴露于光线下。</li>
	</ol>
</li>
</ol>5. PN 注射
<ol><li>在整个 PN 注射过程中, 在冰上解冻 Cas9/sgRNA RNP, 并在冰上保持复合体。</li>
	<li>在注射实验前立即用 Cas9/sgRNA RNP 溶液加载注射针。将注射针的后端放入含有 Cas9/sgRNA RNP 溶液的管内, 并允许 Cas9/sgRNA RNP 溶液通过毛细管作用填充针。</li>
	<li>准备注射碟和针头。将持有者的吸管与矿物油填充到3毫米的支架针的弯曲处。将支架放在微量中, 将针的尖端水平放在盘子的底部, 然后用吸管将针的尖端填充到中间。设置注射针在 10-15 角度对面的持有人。</li>
	<li>PN 注射液<ol>
<li>确定一个合子, 并应用一个良好的吸力与持有人针。理想情况下, 男性和女性核在相同的焦距范围内, 并与保持架平行。</li>
		<li>穿透透明和男性核 (3 点的位置) 与注射针。</li>
		<li>一旦原膨胀, 将注射针迅速取出, 防止细胞核附着在针头上, 避免损伤原。</li>
	</ol>
</li>
</ol>6. 受精卵转移到 Pseudopregnant 仓鼠
<ol><li>麻醉 pseudopregnant 仓鼠 (与上述输精管结扎术中描述的相同), 并将其放置在腹卧床的外科悬垂或干纸巾上。</li>
	<li>将眼部润滑剂应用于动物眼睛, 防止眼部干燥, 用布或纸遮住头部以保护眼睛免受光线的影响。准备身体的侧面, 剃须在身体两侧的4厘米 x 4 厘米的皮肤面积, 然后应用3次聚维酮碘磨砂和3倍70% 乙醇磨砂。使2厘米垂直切口2厘米尾端到最后一个肋骨 (如图 4a, 4b) 在动物背部的两侧。</li>
	<li>用钳子抓住脂肪垫, 轻轻地拉, 直到输卵管和子宫可见。用止血夹住脂肪垫, 并在脊柱上反射脂肪垫背 (图 4c)。定位生殖道, 使子宫管可以穿透30针的胚胎移植 (图 4d)。</li>
	<li>用 HECM-9 在新菜中冲洗注射受精卵。使用新的玻璃吸管 (直径: 〜 150-200 µm) 负载 10-15 受精卵。将受精卵排列成一连串的珍珠, 接着是几个气泡。将吸管插入6.3 步的开放穿透管中, 轻轻吹入吸管, 将受精卵转移至输卵管。继续吹, 直到第一个气泡被释放到输卵管 (图 4e)。</li>
	<li>释放脂肪垫并将组织返回腹部。在相同的过程中, 将相同数量 (10-15) 的注射胚胎转移到另一个输卵管。</li>
	<li>缝合肌肉组织和皮肤在2层。用盐酸丁丙诺啡 (0.5 毫克/千克) 皮下注射作为术后镇痛。把仓鼠归还笼子, 把笼子放在暖和的垫子上, 以便恢复。当动物完全从麻醉中恢复时, 将笼子交还给动物室。术后镇痛药六小时后再给你。</li>
</ol>

<ol>
	<li>Takahashi, M., Hori, M., Mutoh, M., Wakabayashi, K., Nakagama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+animal+models+of+pancreatic+carcinogenesis+for+prevention+studies+and+their+relevance+to+human+disease.">Experimental animal models of pancreatic carcinogenesis for prevention studies and their relevance to human disease.</a> Cancers (Basel). 3 (1), 582-602 (2011).</li><li>Wold, W. S., Toth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+three--Syrian+hamster+as+an+animal+model+to+study+oncolytic+adenoviruses+and+to+evaluate+the+efficacy+of+antiviral+compounds.">Chapter three--Syrian hamster as an animal model to study oncolytic adenoviruses and to evaluate the efficacy of antiviral compounds.</a> Adv Cancer Res. , 69-92 (2012).</li><li>Thomas, M. A., 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=Syrian+hamster+as+a+permissive+immunocompetent+animal+model+for+the+study+of+oncolytic+adenovirus+vectors.">Syrian hamster as a permissive immunocompetent animal model for the study of oncolytic adenovirus vectors.</a> Cancer Res. 66 (3), 1270-1276 (2006).</li><li>Thomas, M. A., Spencer, J. F., Wold, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+Syrian+hamster+as+an+animal+model+for+oncolytic+adenovirus+vectors.">Use of the Syrian hamster as an animal model for oncolytic adenovirus vectors.</a> Methods Mol Med. , 169-183 (2007).</li><li>Hogarth, C. A., Roy, A., Ebert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+evidence+for+the+absence+of+a+functional+cholesteryl+ester+transfer+protein+gene+in+mice+and+rats.">Genomic evidence for the absence of a functional cholesteryl ester transfer protein gene in mice and rats.</a> Comp Biochem Physiol B Biochem Mol Biol. 135 (2), 219-229 (2003).</li><li>Ebihara, H., 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=A+Syrian+golden+hamster+model+recapitulating+ebola+hemorrhagic+fever.">A Syrian golden hamster model recapitulating ebola hemorrhagic fever.</a> J Infect Dis. 207 (2), 306-318 (2013).</li><li>Jove, M., 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=Lipidomic+and+metabolomic+analyses+reveal+potential+plasma+biomarkers+of+early+atheromatous+plaque+formation+in+hamsters.">Lipidomic and metabolomic analyses reveal potential plasma biomarkers of early atheromatous plaque formation in hamsters.</a> Cardiovasc Res. 97 (4), 642-652 (2013).</li><li>Vairaktaris, E., 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=The+hamster+model+of+sequential+oral+oncogenesis.">The hamster model of sequential oral oncogenesis.</a> Oral Oncol. 44 (4), 315-324 (2008).</li><li>Paciello, O., 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=Syrian+hamster+infected+with+Leishmania+infantum:+a+new+experimental+model+for+inflammatory+myopathies.">Syrian hamster infected with Leishmania infantum: a new experimental model for inflammatory myopathies.</a> Muscle Nerve. 41 (3), 355-361 (2010).</li><li>Safronetz, D., Ebihara, H., Feldmann, H., Hooper, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Syrian+hamster+model+of+hantavirus+pulmonary+syndrome.">The Syrian hamster model of hantavirus pulmonary syndrome.</a> Antiviral Res. 95 (3), 282-292 (2012).</li><li>Fan, Z., 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=Efficient+gene+targeting+in+golden+Syrian+hamsters+by+the+CRISPR/Cas9+system.">Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system.</a> PLoS One. 9 (10), e109755 (2014).</li><li>McKiernan, S. H., Bavister, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+one-cell+hamster+embryos+with+water+soluble+vitamins:+pantothenate+stimulates+blastocyst+production.">Culture of one-cell hamster embryos with water soluble vitamins: pantothenate stimulates blastocyst production.</a> Hum Reprod. 15 (1), 157-164 (2000).</li><li>Takenaka, M., Horiuchi, T., Yanagimachi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+light+on+development+of+mammalian+zygotes.">Effects of light on development of mammalian zygotes.</a> Proc Natl Acad Sci U S A. 104 (36), 14289-14293 (2007).</li></ol>

该公司是一家专门为生物医学研究和农业应用创造基因工程动物的生物技术企业, 在这方面有经济利益。

为了更好地利用黄金叙利亚仓鼠的潜力作为人类疾病的模型, 我们开发了一个 PN 注射协议, 以提供一个 CRISPR/Cas9 复合体为目标的仓鼠基因组。PN 注入协议优化了几个关键变量, 包括胚培养基、温度和光的波长13。还有几个仓鼠特定的动物处理程序, 需要遵循成功地进行基因打靶的仓鼠。例如, 性成熟的雌性仓鼠有稳定的4天的动情周期, 不能与外源激素同步 (例如PMSG)。因此, ?...

金黄叙利亚仓鼠 (Mesocricetus 鲫) 是生物医学研究中使用最广泛的啮齿动物之一。根据美国农业部的数据, 2015年美国使用了大约10万只仓鼠, 这代表了动物福利法 (http://www.aphis.usda.gov) 所涵盖的物种在实验室动物总数中的13%。2017年3月10日)。
在研究一些人类疾病时, 仓鼠比其他啮齿动物提供了一些优势。例如, nitrosobis (2-代丙基) 胺 (防喷) 诱导的胰胆管癌的组织病理学与人胰腺肿瘤相似, 而防喷治疗主要诱发大鼠甲状腺肿瘤和肺、肝肿瘤鼠标1。由于仓鼠是唯一发现支持复制腺的小鼠, 它们也是测试 adenovirus-based 溶载体和 anti-adenovirus 药物的选择模型2,3,4。另一个例子, 其中仓鼠模型提供了比老鼠和老鼠的优势是在高脂血症的研究。人和仓鼠在脂质代谢通路上具有很大的相似性, 两个物种携带胆固醇酯转运蛋白 (CETP) 的基因编码, 这在脂代谢中起着中心作用, 而 CETP 在小鼠和大鼠中则不存在5。此外, 在感染埃博拉病毒6后, 仓鼠发展出血性疾病更能代表人类的表现。仓鼠也是研究动脉粥样硬化的选择模型7, 口腔癌的8, 和炎症病9。最近, 它也已经证明, 仓鼠是高度敏感的安第斯山脉病毒感染和发展汉坦病毒性肺综合征样疾病, 提供了唯一的啮齿动物模型的安第斯山脉感染10。
为了解决新的基因动物模型无法满足的需要, 研究人类疾病, 没有可靠的小鼠模型, 我们最近成功地将 CRISPR/Cas9 系统应用到仓鼠, 并产生了几行基因工程仓鼠11。仓鼠受精卵对环境环境非常敏感, 因此在其他物种中开发的 PN 注射协议是不合适的。因此, 我们开发了一种用于仓鼠的 PN 注射协议, 它适用于处理仓鼠胚胎的特殊要求体外。在这里, 我们描述了详细的 PN 注射过程中使用的 CRISPR/Cas9 系统和相应的步骤, 从单一指南 RNA (sgRNA) 的准备转移到接受雌性注射胚胎。

<table>
	<tbody>
		<tr>
			<td>Cas9</td>
			<td>Invitrogen</td>
			<td>B25640</td>
			<td>1 ug/ul (~6.1 uM)</td>
		</tr>
		<tr>
			<td>GeneArtTM Precision Synthesis Kit</td>
			<td>Invitrogen</td>
			<td>A29377</td>
			<td>For sgRNA synthesis</td>
		</tr>
		<tr>
			<td>Albumin from human serum</td>
			<td>Sigma</td>
			<td>A1653</td>
			<td>For cultivation medium</td>
		</tr>
		<tr>
			<td>Illuminator</td>
			<td>Nikon</td>
			<td>NI-150</td>
			<td>For embryo transfer</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>New Brunswick</td>
			<td>Galaxy 14S</td>
			<td>For embryo cultivation</td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>PB-7</td>
			<td>For making injection needles</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ti-S</td>
			<td>For microinjection</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>invitrogen</td>
			<td>SMZ745T</td>
			<td>For embryo transfer</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M1840</td>
			<td>Keep in dark</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>Sigma</td>
			<td>G4877-2000IU</td>
			<td>For superovulation</td>
		</tr>
	</tbody>
</table>

production of genetically engineered golden syrian hamsters by pronuclear injection of the crispr/cas9 complex

在小鼠体内首次建立了原 (PN) 注射技术, 将国外的遗传物质引入到单细胞阶段胚胎的核中。引入的遗传物质可以融入胚胎基因组, 并在移植后的胚胎转移到寄养母亲之后, 利用外来的遗传信息产生转基因动物。在小鼠成功后, PN 注射液已成功应用于其他许多动物品种。近年来, PN 注射成功地引入了基因修饰的试剂, 如 CRISPR/Cas9 系统, 在几个实验室和家畜物种中实现 site-specific 的基因修饰。除了掌握特殊的显微注射技术, 通过 PN 注射液生产转基因动物外, 研究人员还必须了解目标物种的繁殖生理和行为, 因为每个物种都呈现出独特挑战.例如, 金黄叙利亚仓鼠胚胎有独特的处理要求在体外, 因此 PN 注射技术是不可能的在这个种类直到最近突破由我们的小组。通过我们的物种改良 PN 注射协议, 我们成功地生产了几个基因敲除 (KO) 和敲 (基) 仓鼠, 已成功地用于人类疾病模型。在这里, 我们描述了 PN 注射程序, 交付 CRISPR/Cas9 复合物的受精卵的仓鼠, 胚胎处理条件, 胚胎移植程序, 和畜牧业所需的生产转基因仓鼠。

所述协议在生产转基因仓鼠中的效率取决于以下两个关键步骤的结果: 受赠雌性的活产率和预期基因修饰的活幼崽数。活产率是胚胎质量的直接结果, 是个体进行 PN 注射和胚胎移植过程的技术。为了确保纵胚胎的发育潜能不会受到损害 , 在对仓鼠胚胎进行的体外处理过程中需要非常小心。我们定期获得 60-80% 的 pseudopregnant 受赠女性的活产率。表 3演示了?...

Watch this Scientific Journal Video about Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex at JoVE.com

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

原 (PN) 注射的聚类定期 interspaced 短复发重复 (CRISPR) 和 CRISPR 相关 protein-9 核酸 (CRISPR/Cas9) 系统是一种高效的方法生产基因工程金黄叙利亚仓鼠。在此, 我们描述了详细的 PN 注射协议生产的基因剔除仓鼠与 CRISPR/Cas9 系统。

CRISPR/Cas9 复合原注射液生产基因工程金黄叙利亚仓鼠

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

production-genetically-engineered-golden-syrian-hamsters-pronuclear

Research

JoVE Journal

Bioengineering

12.8K 次观看.  Utah State University. 原 (PN) 注射的聚类定期 interspaced 短复发重复 (CRISPR) 和 CRISPR 相关 protein-9 核酸 (CRISPR/Cas9) 系统是一种高效的方法生产基因工程金黄叙利亚仓鼠。在此, 我们描述了详细的 PN 注射协议生产的基因剔除仓鼠与 CRISPR/Cas9 系统。

视频: Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

基因转染向着文化微模式球体板细胞基因修饰的细胞移植

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

科研

生物工程

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

可注射药物加载超分子凝胶局部导管注入猪心

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was pretreated with NaOH and washed to a neutral pH. Cellulase was added to the pretreated bamboo powder to generate the hydrolysate, which contained 30 g/L glucose and 15 g/L xylose and was used as the carbon source to prepare a medium for chemical production. When cultured in microaerobic conditions, 12.7 g/L 2,3-butanediol was produced by wildtype K. pneumoniae. In aerobic conditions, 13.0 g/L R-acetoin was produced by the budC mutant of K. pneumoniae. A mixture of 25.5 g/L 2-ketogluconic acid and 13.6 g/L xylonic acid was produced by the budA mutant of K. pneumoniae in a two-stage, pH-controlled fermentation with high air supplementation. In the first stage of fermentation, the culture was maintained at a neutral pH; after cell growth, the fermentation proceeded to the second stage, during which the culture was allowed to become acidic.

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bamboo powder was pretreated with NaOH and enzymatically hydrolyzed. The hydrolysate of bamboo was used as the feedstock for 2,3-butanediol, R-acetoin, 2-ketogluconic acid, and xylonic acid production by Klebsiella pneumoniae.

化学品生产<em&gt;肺炎克雷伯菌</em使用竹水合物作为原料

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

对西方玉米 Rootworm、 Diabrotica virgifera virgifera、生殖转化胚胎或 CRISPR/Cas9 基因组编辑的显微注射

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

使用模块化金门克隆的多基因结构快速组装

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...