Name: development of leishmania species strains with constitutive expression of egfp
Uploaded: 2023-04-21T13:30:00
Description: Watch this Scientific Journal Video about Development of Leishmania Species Strains with Constitutive Expression of eGFP at JoVE.com

这项工作由巴拿马国家科学、技术和创新秘书处（SENACYT）资助，批准号为NI-177-2016，巴拿马国家研究系统（SNI），授权号为SNI-169-2018，SNI-008-2022和SNI-060-2022。

为了保持样本无菌，所有涉及寄生虫培养的步骤都应在生物安全2级（BSL-2）罩内或根据当地健康和安全法规进行。该协议的图形摘要可在 图1中找到。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64939/64939fig01.jpg"> 图 1：使用 pLEXSY-2.1 载体家族生成表达 eGFP 的 L. panamensis 和 L. donovani</...

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已经在几种原生动物寄生虫中研究了各种报告基因的优缺点。其中，GFP和eGFP具有固有荧光，易于定量和成像。这些蛋白质的荧光活性可以使用荧光显微镜、荧光测定法或流式细胞术进行最少的操作来检测。很少有研究用于产生表达GFP的L。（Viannia）菌株，尽管GFP及其衍生物作为旧世界利什曼原虫物种15,16,18,24</su...

利什曼原虫属的原生动物寄生虫引起利什曼病，这是一种具有广泛临床表现的疾病。该病在98个国家流行，年发病率估计为90万至160万例1。对人类具有致病性的利什曼原虫分为两个亚属，即L。（利什曼原虫）和L.（维亚尼亚）。感染属于L的一些物种。（利什曼原虫）亚属，如多诺瓦尼乳杆菌和婴儿利什曼病，可能导致内脏利什曼病（VL），如果不及时治疗，这是致命的2。属于L的物种。（Viannia）亚属与中美洲和南美洲的大多数皮肤利什曼病（CL）和皮肤黏膜利什曼病（MCL）病例有关，特别是在巴拿马、哥伦比亚和哥斯达黎加，帕纳美军团菌是这些临床表现的主要病因3,4。
现有的抗利什曼原虫化疗包括五价锑、米替福辛和两性霉素 B 等药物，毒性高且价格昂贵。此外，近几十年来耐药性的增加已成为干扰全球患者有效治疗的因素5。利什曼原虫属物种之间在药物敏感性方面存在实质性差异，特别是在新世界和旧世界物种之间 6,7。由于这些原因，有必要努力确定和开发新的抗利什曼原虫药物，特别注意针对特定物种的方法。使用传统方法研究大型候选药物库非常困难，因为这些方法对于评估化合物对细胞内无鞭毛体的活性或进行体内实验非常费力8;因此，有必要开发减少这些缺点的新技术，包括报告基因的实施和高内涵表型筛选测定的开发9。
报告基因的使用已经显示出提高药物筛选过程效率的潜力，因为它促进了高通量和体内测定的开发。表达几种报告基因的重组利什曼原虫寄生虫已由各种研究小组产生。报告基因，如β-半乳糖苷酶、β-内酰胺酶和荧光素酶，已被引入几种利什曼原虫物种，使用游离体载体，显示出在寄生虫的细胞外和细胞内形式的药物筛选中的效用有限 10,11,12,13,14,15.这些方法的局限性在于需要在培养中具有强大的选择压力以避免消除游离体构建体，以及使用额外的试剂来揭示报告基因的活性。相反，绿色荧光蛋白（GFP）及其变体增强型绿色荧光蛋白（eGFP）已被用于产生大量转基因利什曼原虫菌株，用于体外药物筛选测定，因为它们具有灵活性和灵敏度，以及使用流式细胞术或荧光测定法自动化筛选过程的可能性15，16,17,18,19.尽管结果有希望，但这些转基因菌株的培养物在荧光水平上具有高度异质性，因为所有寄生虫中GFP基因的拷贝数并不相同。此外，保持荧光需要对培养物中的寄生虫施加恒定的选择压力，因为GFP基因是在游离体构建体中引入的。
由于上述原因，许多努力都集中在开发生产稳定重组菌株的新方法上。这些努力主要依赖于将报告基因整合到核糖体位点中，利用核糖体基因的较高转录率20。已经产生了婴儿乳杆菌和亚马逊乳杆菌菌株，整合了编码β-半乳糖杀灭酶21、IFP 1.4、iRFP22 和 tdTomato23 的基因，并且已经评估了它们在药物筛选测定中的有用性。不同的小组已经开发出多诺瓦尼乳杆菌菌株，通过同源重组将其编码基因整合到18S核糖体RNA位点（ssu位点）中，从而形成表达GFP;它们在转染的人群中显示出稳定和均匀的GFP表达，包括细胞内无鞭毛体24,25，并且它们在药物筛选测定中成功实施24,25,26。Bolhassani等人27开发了表达GFP的重杆菌和婴儿乳杆菌菌株作为整合的转基因。他们使用了整合载体pLEXSY，最初设计用于在使用L. tarentoale作为宿主的系统中蛋白质的转基因表达28。pLEXSY-GFP载体已被证明对于生成组成型表达GFP 24,25,27,29,30的不同利什曼原虫菌株非常有效。在这些寄生虫中，荧光是均匀的，并以细胞内形式维持，能够在感染小鼠的脚垫病变中检测到27。
在这项工作中，我们描述了用于生成L. panamensis 和 L. donovani 菌株的方法，这些菌株使用pLEXSY系统将编码eGFP的基因作为整合的转基因。通过此过程产生的菌株在我们的实验室中用于进行药物筛选测定，以评估天然和合成来源分子的潜在抗利什曼原虫活性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 Well Microplates</td>
			<td>Corning</td>
			<td>CLS3340</td>
			<td>Flat bottom clear, black polystyrene, sterile, lid</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A4718</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A8351</td>
			<td>BioXtra, suitable for cell culture</td>
		</tr>
		<tr>
			<td>BglII restriction enzyme</td>
			<td>New England BioLabs</td>
			<td>R0144S</td>
			<td>2,000 units. 10,000 units/mL</td>
		</tr>
		<tr>
			<td>Cell Culture Flasks</td>
			<td>Corning</td>
			<td>CLS430168</td>
			<td>Surface area 25 cm2, canted neck, cap (plug seal)</td>
		</tr>
		<tr>
			<td>ChemiDoc Imaging System</td>
			<td>Bio-Rad</td>
			<td>17001401</td>
			<td></td>
		</tr>
		<tr>
			<td>CyFlow Space</td>
			<td>Sysmex</td>
			<td>Not available</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td>powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, &#8805;99.5%</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Loading Buffer</td>
			<td>Sigma-Aldrich</td>
			<td>G2526</td>
			<td>&#160;The rate of migration varies with gel composition. Dilute 1:3 to 1:6 with sample before loading.</td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell Electroporation System</td>
			<td>Bio-Rad</td>
			<td>1652660</td>
			<td>The system is composed of a main unit, two accessory modules, the capacitance extender (CE module) and the pulse controller (PC module), and a ShockPod cuvette chamber.</td>
		</tr>
		<tr>
			<td>Gene Pulser/MicroPulser Electroporation Cuvettes</td>
			<td>Bio-Rad</td>
			<td>1652086</td>
			<td>Pkg of 50, 0.2 cm&#8211;gap sterile electroporation cuvette, for use with the Gene Pulser and MicroPulser Systems, for mammalian and other eukaryotic cells</td>
		</tr>
		<tr>
			<td>Gentamicin solution</td>
			<td>Sigma-Aldrich</td>
			<td>G1397</td>
			<td>50 mg/mL in deionized water, liquid, 0.1 &#956;m filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>GoTaq Long PCR Master Mix</td>
			<td>Promega</td>
			<td>M4021</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES solution</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td>1 M, pH 7.0-7.6, sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>IXplore Standard</td>
			<td></td>
		</tr>
		<tr>
			<td>KpnI-HF restriction enzyme</td>
			<td>New England BioLabs</td>
			<td>R3142S</td>
			<td>4,000 units. 20,000 units/mL</td>
		</tr>
		<tr>
			<td>LB Broth with agar</td>
			<td>Sigma-Aldrich</td>
			<td>L3147</td>
			<td>Highly-referenced nutrient-rich microbial growth powder medium with Agar, suitable for regular&#160;E.coli&#160;culture.</td>
		</tr>
		<tr>
			<td>LB Broth&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>L2542</td>
			<td>Liquid microbial growth medium</td>
		</tr>
		<tr>
			<td>Mini-Sub Cell GT Horizontal Electrophoresis System</td>
			<td>Bio-Rad</td>
			<td>1640300</td>
			<td>Mini horizontal electrophoresis system, includes 8- and 15-well combs, 7 cm x 10 cm UV-transparent tray</td>
		</tr>
		<tr>
			<td>pEGFP-N1-1x</td>
			<td>Addgene</td>
			<td>172281</td>
			<td>Expressing eGFP mRNA fused with 1 tandem repeat of a 50-base sequence</td>
		</tr>
		<tr>
			<td>pLEXSYcon2.1 expression kit</td>
			<td>Jena Bioscience</td>
			<td>EGE-1310sat</td>
			<td>Contains integrative constitutive expression vector pLEXSY-sat2.1. Antibiotic selection of transfectants with Nourseothricin (NTC, clonNAT). Contains all primers for diagnostic PCRs and sequencing.</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Millipore</td>
			<td>529552</td>
			<td>Molecular Biology Grade - CAS 7447-40-7 - Calbiochem</td>
		</tr>
		<tr>
			<td>PureYield Plasmid Miniprep System</td>
			<td>Promega</td>
			<td>A1222</td>
			<td>Up to 15 &#956;g of Transfection-Ready Plasmid from 3 mL cultures.</td>
		</tr>
		<tr>
			<td>Schneider&#8242;s Insect Medium</td>
			<td>Sigma-Aldrich</td>
			<td>S0146</td>
			<td>Medium used in our laboratory for culturing Leishmania.</td>
		</tr>
		<tr>
			<td>SOC Medium</td>
			<td>Sigma-Aldrich</td>
			<td>S1797</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td>for molecular biology, DNase, RNase, and protease, none detected, &#8805;99% (titration)</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>RDD038</td>
			<td>BioReagent, suitable for cell culture, suitable for insect cell culture, &#8805;99.0%, free-flowing, Redi-Dri</td>
		</tr>
		<tr>
			<td>SwaI restriction enzyme</td>
			<td>New England BioLabs</td>
			<td>R0604S</td>
			<td>2,000 units. 10,000 units/mL</td>
		</tr>
		<tr>
			<td>Syringe filters</td>
			<td>Corning</td>
			<td>CLS431212</td>
			<td>regenerated cellulose membrane, diam. 4&#160;mm, pore size 0.2&#160;&#956;m</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td>Thermal cycler system, includes 96-well thermal cycler, power cord, tube support ring</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Promega</td>
			<td>M1801</td>
			<td>Joins two DNA strands with cohesive or blunt ends</td>
		</tr>
		<tr>
			<td>Tris-Borate-EDTA buffer</td>
			<td>Sigma-Aldrich</td>
			<td>T4415</td>
			<td>BioReagent, suitable for electrophoresis, 10&#215; concentrate</td>
		</tr>
		<tr>
			<td>Wizard Genomic DNA Purification Kit</td>
			<td>Promega</td>
			<td>A1120</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard SV Gel and PCR Clean-Up System</td>
			<td>Promega</td>
			<td>A9285</td>
			<td></td>
		</tr>
		<tr>
			<td>XL10-Gold Ultracompetent Cells</td>
			<td>Agilent</td>
			<td>200317</td>
			<td>XL10-Gold Kanr Ultracompetent Cells, 10 x 0.1 mL. Features the kanamycin-resistance gene on the F&#39; episome, for extremely demanding cloning in chloramphenicol-resistant vectors. Efficiency: &#62; 5 x 10 9 transformants/&#181;g pUC18 DNA.</td>
		</tr>
	</tbody>
</table>

development of leishmania species strains with constitutive expression of egfp

利什曼原虫属的原生动物寄生 虫引起利什 曼病，这是一种具有不同临床表现的疾病，影响着全世界数百万人。 多诺瓦尼乳杆菌 感染可导致致命的内脏疾病。在巴拿马、哥伦比亚和哥斯达黎加， 帕纳梅斯乳杆菌 是大多数报告的皮肤和皮肤黏膜利什曼病病例的原因。使用迄今为止可用的方法研究大量候选药物是相当困难的，因为它们对于评估化合物对寄生虫细胞内形式的活性或进行 体内 测定非常费力。在这项工作中，我们描述了 L. panamensis 和 L. donovani 菌株的产生，其基因的组成性表达编码增强的绿色荧光蛋白（eGFP）整合到编码18S rRNA（ssu）的位点中。编码eGFP的基因是从商业载体中获得的，并通过聚合酶链反应（PCR）扩增以富集它并为 BglII和 KpnI酶添加限制性位点。通过琼脂糖凝胶纯化分离eGFP扩增子，用 酶BglII和 KpnI消化，并连接到先前用同一组酶消化的 利什曼原虫 表达载体pLEXSY-sat2.1中。将具有克隆基因的表达载体在 大肠杆菌中繁殖，纯化，并通过集落PCR验证插入片段的存在。纯化的质粒被线性化并用于转染 多诺瓦尼乳杆菌 和 白曲霉 寄生虫。通过PCR验证了该基因的整合。通过流式细胞术评估eGFP基因的表达。通过限制稀释法克隆荧光寄生虫，并使用流式细胞术选择荧光强度最高的克隆。

在构建pLEXSY-eGFP构建体并转化感受态 大肠杆菌 细胞后，含有带有eGFP插入片段的构建体的菌落将在运行第1.2节所述的集落PCR后产生约859 bp产物（图3A）。使用 SwaI从阳性菌落中纯化的质粒的全消化应在凝胶电泳中得到两个特征片段，一个2.9 kbp片段，是PLEXSY载体的一部分，其中包含在 大肠杆菌中复制和选择的所有必要元件，以及一个7-8 kpb片段代表用于转染?...

Watch this Scientific Journal Video about Development of Leishmania Species Strains with Constitutive Expression of eGFP at JoVE.com

Development of Leishmania Species Strains with Constitutive Expression of eGFP

在这里，我们描述了用于生成使用pLEXSY系统将eGFP基因表达为稳定整合转基因的L. panamensis 和 L. donovani 菌株的方法。通过限制稀释来克隆转染的寄生虫，并选择两种物种中荧光强度最高的克隆进一步用于药物筛选测定。

具有eGFP组成型表达的 利什曼原虫 菌株的发展

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people ...

该协议是生成可用于高通量筛选测定以评估分子潜力和传统活性的菌株的关键 展示该程序的将是我实验室的研究助理Juana Quintero和Laura Pineda。首先，使用高保真聚合酶扩增靶基因以保留编码序列。将引物以0.2微摩尔的终浓度添加到反应混合物中，并按照手稿中所述运行PCR循环方案。使用常规PCR产物纯化试剂盒纯化扩增的PCR片段。要修剪eGFP PCR产物的末端，请根据制造商的说明与Kpn I酶进行反应，并在37摄氏度下孵育一小时。然后在反应中加入100毫摩尔氯化钠和10单位Bgl II，孵育15分钟。消化后，如前所示纯化反应。接下来，按照前面所示的顺序酶切，用Bgl II和Kpl I消化pLEXSY表达载体。在含有连接酶缓冲液和三个单位 T4 DNA 连接酶的 20 μL 反应中连接 100 纳克消化的 pLEXSY 载体和 28 纳克消化的 eGFP。在四摄氏度下孵育过夜。在孵育结束时，使用标准转化方案用连接产物转化感受态大肠杆菌细胞。将转化的细胞接种在LB氨苄青霉素琼脂中，并在30摄氏度下孵育24小时以选择重组克隆。筛选菌落后，从阳性克隆中纯化质粒DNA，以便使用商业质粒分离试剂盒进行后续转染。为了产生质粒的线性化片段，取至少10微克纯化的pLEXSY eGFP质粒，并在25摄氏度下用SWA1消化三到四个小时。在孵育结束时，在65摄氏度下加热消化产物20分钟。然后在琼脂糖凝胶电泳中运行消化产物以分离所得片段。根据制造商的说明，使用商业琼脂糖凝胶提取试剂盒纯化表达盒。培养寄生虫培养物，直到有足够的对数期或早期固定期前鞭毛体。如果需要，合并多个培养瓶的内容物。在室温下以2， 000G离心寄生虫培养物三分钟。将沉淀重悬于4摄氏度的电穿孔缓冲液中，以获得每毫升10至8个寄生虫的浓度，并将其放在冰上10分钟。同时，在 pH 8.0 的 50 微升水或 10 毫摩尔三缓冲液和直径为 2 毫米的电穿孔比色皿中构建具有 2 至 10 微克线性化 pLEXSY eGFP 的预冷管。接下来，将350微升预冷的寄生虫加入带有线性质粒的管中，并将整个400微升转移到冰上的电穿孔比色皿中。用质粒电穿孔寄生细胞，同时用没有质粒DNA的寄生虫细胞作为阴性对照。将比色皿放在冰上 10 分钟。将电穿孔寄生虫转移到5毫升适当的培养基中，并在26摄氏度下孵育20小时。电穿孔后约20小时，在显微镜下观察培养物，并根据所使用的pLEXSY质粒添加适当的选择性抗生素。显微镜下跟踪培养物，直到有和没有质粒DNA电穿孔的寄生虫有明显差异。为了通过流式细胞术验证寄生虫荧光，在室温下以2， 000G离心一毫升固定相培养物三分钟。在PBS中洗涤细胞两次后，将最终沉淀重悬于一毫升PBS中。运行样本并以每秒 0.5 微升的速度收集 20， 000 个事件。将前向散射、侧向散射和荧光一通道的增益值分别设置为 225.0、200.0 和 520.0。创建一个包含寄生虫种群的门 G1。通过该门过滤FL1通道以确定前鞭毛体的自发荧光，并为FL1通道设置范围门G2。运行转染的寄生虫以验证是否有荧光。记录G1中荧光寄生虫的百分比和G2中荧光强度的平均值。通过常规苯酚氯仿提取或商业试剂盒从两到五毫升固定相寄生虫培养物中纯化基因组DNA。使用手稿中所述的循环方案对pLEXSY-sat2.1进行诊断PCR。通过流式细胞术确认荧光后，制备浓度为每毫升五个前鞭毛的重组寄生虫稀释液。将100微升稀释液加入96孔板的每个孔中，并使板不受干扰至少12小时。以最小100X的放大倍率显微镜跟踪板，直到检测到寄生虫生长的孔。当井中充满寄生虫时，使用内容物接种五毫升培养物。一旦克隆接种的培养物到达固定相，如前所述使用流式细胞术验证荧光。选择具有98%至99%荧光寄生虫和最高平均荧光强度的克隆进行体外和体内测定。用pLEXSY eGFP构建体转化的大肠杆菌细胞的集落PCR产生了859碱基对产物。使用SWA1对质粒进行全酶切得到两个片段，一个2.9千碱基对片段，其中包含在大肠杆菌中复制和选择所需的所有元件，另一个7至8千碱基对片段代表用于转染的线性表达盒。流式细胞术分析显示，82%的转染的帕纳梅斯乳杆菌寄生虫表达eGFP。多克隆选择后，流式细胞术分析的多诺瓦尼乳杆菌寄生虫中有98.44%是荧光的，而帕纳梅氏乳杆菌的荧光率为82.0%。pLEXSY eGFP成功整合到SSU基因座中，在诊断PCR中产生了2.3千碱基对产物。选择具有最高荧光寄生虫百分比和平均荧光强度的克隆进一步用于药物筛选测定。该方法允许用

Research

JoVE Journal

Genetics

Development of Leishmania Species Strains with Constitutive Expression of eGFP

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

科研

遗传学

Generation of Fluorescent Protein Fusions in Candida Species

Generation of Fluorescent Protein Fusions in Candida Species

在荧光蛋白融合的一代<em&gt;念珠菌</em&gt;物种

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

无基因克隆的基因破坏的变形链球菌菌株的生成

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

血管动力尾静脉注射对小鼠肝细胞基因的本构和诱导系统的修饰

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

提高吞吐量的轰击大麦糊粉层层中基因表达的测定

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

一种基于昆虫蜕皮激素受体的奇异基因开关, 用于对转基因进行有鲁棒性、可逆性和可忽略的泄漏的刻意表达

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

工程神经前体宫内移植,用于Vivo神经结构研究

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

通过全基因组互惠性分析,对糖母菌菌种间热耐性差异的遗传图谱

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

具有eGFP组成型表达的 利什曼原虫 菌株的发展

具有eGFP组成型表达的利什曼原虫菌株的发展