这项工作得到了干杯研究所的支持 (计划 Estatal de i + 2013-2016): 赠款 CP15-00012、PI16-00860 和 CIBER (CB06/02/0053), 由欧洲发展区域基金共同出资 "实现欧洲的一种方式" (ERDF)。talento (2016-T1/生物-1105) 和西班牙 Ministerio Economía、工业 y Competitividad (BIO2017-85056-P) 的 Excelencia, 得到了马德里地区政府的 Atracción de ( ) 计划的支持。ASM 是由由欧洲社会基金 "投资于你的未来" (Servet) 和 ERDF 共同出资的干杯 (MS15/00012) 的一支米格尔的奖学金。

1. 抗生素耐药性基因实验系统的构建
注: 以大肠杆菌MG1655 作为质粒或染色体编码抗生素耐药性基因的受体菌株。抗生素耐药性基因编码在染色体或多拷贝产质粒中, 否则等基因系应变(图 1)。
<ol><li>
在λ噬菌体集成站点 (attB)20的 MG1655 染色体中插入耐药性基因。
	<ol>
<li>通过 PCR 方法放大染色体attB站点两侧的500个 bp 长区域。使用寡核苷酸 YbhC (5 '-CCTGTACCGTACAGAGTAAT-3 ') 和 attB (5 '-GCCCGCCACCCTCCGGGCCGGTATAAAAAAGCAGGCTTCA-3 ') 为左同源区和 attB F (5 '-AGCGCCCTAGCGCCCGCTCCTTATACTAACTTGAGCGAAA-3 ') 和 YbhB/R (5 '-TGGCGATAATATTTCACCGC-3 ') 为正确的一个。使用引物 Tem1-pBAD-F (5 '-TGAAGCCTGCTTTTTTATACCGGCCCGGAGGGTGGCGGGC-3 ') 和 Tem1-pBAD-R (5 '-TTTCGCTCAAGTTAGTATAAGGAGCGGGCGCTAGGGCGCT-3 ') 放大血乳酸TEM-1抗性基因。设计底漆与大约 20 bp (在 5 ' 末端) 序列显示互补到将被熔化的片断。</li>
		<li>将同源区与抗生素耐药性基因 PCR 产品 (包括其启动子区域) 结合使用等温组件21, 在50摄氏度为30分钟。</li>
		<li>Electroporate MG1655 细胞含有质粒 pKOBEG。 注: pKOBEG 是一种热敏载体, 含有λ红色机械, 促进染色体和 PCR 产品之间的同源性等位基因交换22。<ol>
<li>使用1µL 的产品从步骤 1.1.223到40µL electrocompetent 细胞在2毫米小试管在4°c 和2.5 伏。并用重悬细胞在1毫升 LB 汤 + 0.2% 阿拉伯糖, 以保持λ红色机械的表达。</li>
			<li>将总容积转移到离心管, 在30摄氏度 (允许温度为 pKOBEG) 上孵化2小时, 并进行剧烈晃动 (在一个轨道振动筛中 200 rpm), 以便在染色体中插入电阻标记的表型表达式。</li>
			<li>在 LB 琼脂上板上含有适当抗生素的细胞, 用于选择抗生素耐药性基因 (氨苄西林或羧苄青霉素100毫克/升用于血乳酸 TEM-1). </li>
		</ol>
</li>
		<li>板100µL 在一个培养皿, 并旋转下来的其余的体积, 并用重悬它在100µL 新鲜的 LB 汤, 并在另一培养皿盘子。孵化过夜在42摄氏度。 注: 此温度不允许复制 pKOBEG。能够在这些条件下生长的菌落将失去 pKOBEG, 并将抵抗基因集成到attB站点中 (为了简单起见, 这一菌株将被称为MG1655:瑞莎从此处起)。</li>
		<li>通过用氯霉素 (pKOBEG 含有抗性标记的抗生素) 复制板上的兴趣菌落来测试 pKOBEG 的损失。 注: 在30ºC 的许可温度下, 氯霉素板缺乏生长, 表明质粒缺乏。</li>
		<li>为了验证克隆, PCR 放大结构使用外部引物 YbhC (5 '-TTTGTGACCAGAAGACCGCA-3 ') 和 YbhB-外部 (5 '-CTCATCAGTAACGATCTGCG-3 '), 通过凝胶电泳验证扩增子的正确大小和序列的纯化的产品, 以确保插入基因的顺序是正确的。</li>
	</ol>
</li>
	<li>
在多拷贝产质粒中插入耐药性基因。 注: 由于具有极高拷贝数的质粒倾向于大量减少细菌宿主的适用性, 我们建议使用具有自然复制起源的多拷贝产质粒, 如 p3655 (pSU18T-pBAD gfp2, ColE1-type 起源于复制24)。这些质粒通常为每个细胞提供大约15-20 个拷贝的适度拷贝数。<ol>
<li>pcr 放大选择的抗生素耐药性基因 (包括启动子区), phosphorylate pcr 产物, 并将其结扎到 pcr 扩增的质粒:<ol>
<li>PCR 放大抗生素耐药性基因;对于乳酸TEM-1使用底漆 Tem1-pBAD-F (5 '-TGAAGCCTGCTTTTTTATACCGGCCCGGAGGGTGGCGGGC-3 ') 和 Tem1-pBAD-R (5 '-TTTCGCTCAAGTTAGTATAAGGAGCGGGCGCTAGGGCGCT-3 ')。</li>
			<li>Phosphorylate 的纯化产品使用 T4 核苷酸激酶, 按照制造商的指示。</li>
			<li>应用高保真聚合酶和寡核苷酸 pBAD (5 '-CGTTGATCGGCACGTAAGAG-3 ') 和 pBAD R (5 '-AAACGACGGCCAGTGCCAAG-3 '), PCR 扩增质粒骨干。</li>
			<li>结扎两个 PCR 片段使用 T4-ligase 以下制造商的指导方针。</li>
		</ol>
</li>
		<li>Electroporate, 如前所述, 40 µL 的 electrocompetent大肠杆菌DH5-α细胞最多1µL 结扎产品, 并选择适当的抗生素耐药性基因 (氨苄西林或羧苄青霉素在100毫克/升用于血乳酸TEM-1) 和质粒主干。 注: 补充磅与阿拉伯糖这里是不必要的。</li>
		<li>使用读者选择的商业迷你准备套件提取质粒:<ol>
<li>在室温下, 通过离心在 1.2万 x g 上收获5毫升的隔夜文化。并用重悬250µL 泥沙溶液中的细胞, 混合均匀。加入250µL 的溶解液, 混合均匀。添加350µL 中和, 混合良好。</li>
			<li>将上清液转移到所提供的 DNA 结合柱, 离心机1分钟, 并丢弃流经。洗两次柱与500µL 的洗涤液, 离心一分钟, 并丢弃 flowthrough 在每次洗涤。离心空列1余分钟, 以除去残留洗涤缓冲器。</li>
			<li>把柱子放在干净的管子里, 将50µL 的超纯净水添加到膜中, 洗脱纯化的 DNA。离心机为1-2 分钟, 收集含有纯化质粒的流动。利用插入序列外的引物对质粒中感兴趣的基因进行序列化, 以确认序列是正确的, 在进化实验之前, 抗性基因中没有突变存在。</li>
		</ol>
</li>
		<li>一旦序列被证实, electroporate 的质粒在大肠杆菌MG1655 菌株, 如上文所述。 注意: 这会产生应变MG1655/pRESA.
</li>
	</ol>
</li>
</ol>2. 进化拯救方法以实验方式发展抗生素耐药性 (图 1)
<ol><li>对 LB 板研究中的菌株进行条纹: MG1655::瑞莎和 MG1655/pRESA 加上敏感的父母应变, MG1655。 注: 质粒 pRESA 应稳定在 MG1655, 所以没有必要添加抗生素的 LB 板。在大肠杆菌中的突变率足够低, 因此一旦验证了构造, 就不必在每一步验证质粒序列。</li>
	<li>在每个井中准备96井板和200µL 磅, 并接种独立井每株48个独立的菌落 (每株一张)。在37摄氏度和200转每晚孵化板块。对这些创始人种群保持冻结的库存。 注: 为防止和控制96井板中的文化交叉污染, 在整个96井板上使用无菌培养基的锂接种井进行棋盘板设计。在整个实验过程中使用这个板块设计。</li>
	<li>启动进化救援实验, 接种2µL 从板块的每个井与创建者人口在新的96井板材与198µL 磅在每个井与抗生素的亚抑制浓度在研究中。为进化抢救方法开始与¼或1/8 最小的抑制浓度 (麦克风, 确定以前的25) 的抗生素在 LB 为每三株和双倍的浓度的抗生素每日。使用此方法可以最大限度地提高种群获取电阻突变的几率26。孵化的板块在37摄氏度和 200 rpm 20 小时。 注: 测量创始人群体的隔夜 OD。如果菌株之间的 OD 有显著的差异, 正确的初始接种, 以相同数量的细胞每井开始实验。</li>
	<li>每天, 衡量每一个人口的 OD, 在隔夜文化和执行转移的文化, 如2.3 所述, 到新的96井板与双倍的抗生素浓度比前一天。在37摄氏度和200转每分钟孵化20小时的板材。 注: 1:100 稀释因子每天产生大约6-7 代。</li>
	<li>同时, 在2.3 和2.4 所述的相同条件下, 传播每个菌株的控制种群, 但缺乏抗生素。 注意: 控制人群将有助于区分由于抗生素的存在和一般突变帮助细菌适应实验条件而产生的突变。在缺乏抗生素的情况下发生的平行突变可能有助于细菌适应实验条件, 而与抗生素耐药性无关。</li>
	<li>通过用平板阅读器测量600毫微米 (OD) 的吸收量, 每天跟踪幸存人口的数量。 注: 光密度值低于0.1 表示种群的灭绝。有关生存曲线的示例, 请参见图 2 。</li>
	<li>定期 (每3-5 天) 保存所有人口的冰冻库存。</li>
	<li>使用日志秩测试 [RStudio (版本 0.99.486) 中的 "包" 生存期], 以确定在抗生素浓度不断增加的情况下, 不同菌株的种群存活率的统计差异12。 注: 本实验将确定多拷贝产质粒是否事实对特定抗生素和基因研究的抗生素耐药性的演变。</li>
</ol>3. 抗生素耐药性演变的分子基础 (图 1)
<ol><li>执行总 DNA 提取从代表性数量的人口和克隆跨菌株和治疗。包括父母菌株 (MG1655、MG1655:瑞莎和 MG1655/pRESA), 以便能够检测在实验过程中积累的突变。 注: 有关 DNA 提取套件的示例, 请参阅材料表。每个套件都有不同的协议;遵循制造商的指示。</li>
	<li>量化 DNA 质量和浓度。有不同的方法来确定 DNA 的质量和浓度。测定吸收率 260 nm/280 nm 和 260 nm/230 nm 的质量。使用荧光, DNA 结合染料测量 dna 浓度后, 制造商的指示, 和琼脂糖凝胶电泳, 以确认没有 DNA 降解或 RNA 污染。</li>
	<li>利用来自全种群和个体克隆样本的深 DNA 测序, 研究抗生素耐药性的遗传基础。 注: 我们在英国牛津大学人类遗传学信托中心进行了所有的测序。有关详细信息, 请参阅 San 文澜et 。201612。</li>
	<li>使用 breseq 0.26.1 管线27、28检测突变, 使用多态性模式估计种群中突变的频率。将不同的进化基因组与父母基因组进行比较, 以检测在实验中积累的突变。</li>
	<li>比较在对照人群中积累的突变与在抗生素浓度不断增加的人群中的变化, 以区分帮助细菌适应一般实验条件的突变 (在控制人群中发现的那些) 和那些涉及抗生素耐药性的人 (那些仅在受到抗生素压力的人群中发现)。 注意: 平行突变的数量通常是低的, 所以治疗之间的不同的平行突变可以很容易地评估。除了研究中的抗生素耐药性基因突变外, 它很可能在染色体中发现与抗生素耐药性相关的其他突变, 例如孔蛋白或排便系统中的这种突变 12.</li>
	<li>采用与本议定书1节相同的方法, 重建父母菌株中抗生素耐药性基因的突变。按照《议定书》1.1 和1.2 的规定, 在父母背景中引入新的变异 (使用进化后的基因作为 PCR 模板)。分析这些新结构的耐药性表型, 以确定突变的作用。</li>
</ol>

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作者没有什么可透露的。

我们提出了一个新的协议, 结合分子生物学, 实验进化和深 DNA 测序, 旨在研究多拷贝产质粒在细菌耐药性进化中的作用。虽然该协议结合了不同领域的技术, 但开发它所需的所有方法都很简单, 可以在常规微生物学实验室中进行。该协议中最关键的步骤可能是建立模型系统菌株和重建实验后观察到的突变 (使用完全相同的方法进行)。然而, 等温装配系统21, 大大简化了此协议, 因此任何具有中级经验的分子生物学的用户都可以实现它。
该议定书的另一个关键步骤是在不断增加的抗生素浓度下的实验性进化。作为一个例子, 这个协议开始实验与¼1/8 的麦克风的菌株, 然后加倍的浓度抗生素每天。然而, 较低的抗生素变化率可能会增加进化拯救从灭绝的机会26。因此, 抗生素浓度的变化率是可以改变的参数之一, 以促进抗生素耐药性的演变。
DNA 测序和分析也是实验设计的关键方面。在实验中不同的时间点, 对来自整个种群和单个克隆的 DNA 样本进行测序, 结果更直接。从人群中测序结果将揭示治疗中突变剖面的一般差异, 以及选择性地扫描随时间推移的有益突变和克隆干扰的潜在事件。在分析种群中的序列时, 最好筛选出任何人口中从未超过10% 频率的突变。单个克隆的序列有助于确认从人群中获得的结果, 最重要的是揭示了在人口水平上观察到的不同突变之间的具体组合。这些特定的关联可能有助于揭示突变之间的上位相互作用, 这在细菌适应31中起着关键作用。
利用这种方法, 我们最近发现, 多拷贝产质粒加速了抗生素耐药性的进化, 首先是通过增加新突变的出现率, 然后通过放大基因剂量增加的突变效应12.因此, 我们开发的方法作为一个工具来调查抗生素耐药性的演变, 但它可能有一个更广泛的应用范围。也就是说, 这个系统可以用来调查任何细菌基因的能力进化到一个新的或改进的功能, 更普遍的方式。例如, 这个系统可以用来测试代谢酶/通路使用新碳基板的能力32。此外, 它可以代替 hypermutators (细胞系统中涉及 DNA 不匹配修复的细菌) 来研究细菌的自适应基因进化, 避免 hypermutators 引入的突变偏倚。

细菌耐药性是一个主要的健康问题1。在基本水平上, 抗生素耐药性在致病细菌中的传播是自然选择的一个简单的例子, 这是自然选择2,3。简而言之, 抗生素的使用会产生抗性菌株的选择。因此, 进化生物学的一个关键问题是了解影响细菌种群对抗生素的抗药性的能力的因素。选择实验已经成为一个非常强大的工具来调查细菌的进化生物学, 这个领域已经产生了令人难以置信的洞察到广泛的进化问题4,5,6。在实验性进化过程中, 由单亲菌株引发的细菌种群在定义和严格控制的条件下依次传代。在这些文化的生长过程中发生的一些突变增加了细菌的适应性, 这些变化通过自然选择的文化传播。在试验过程中, 种群的样本定期低温保存, 以创建一个非进化的冰冻化石记录。大量的方法可以用来表征不断变化的细菌种群, 但最常见的两种方法是体能测定, 即测量进化细菌与远方祖先竞争的能力, 以及整个基因组测序, 即用于识别驱动适应的遗传变化。经过理查德 Lenski 和同事的开创性工作7,8, 实验进化的标准方法是挑战相对较少数量的复制人口 (通常 < 10), 适应新的环境挑战, 如新的碳源, 温度, 或捕食性的噬菌体。
抗生素耐药性细菌引起的感染成为一个很大的问题, 当抗药性足够高, 不能增加抗生素浓度, 以致命的水平在病人组织。因此, 临床医生对允许细菌进化为高剂量抗生素的耐药性有兴趣, 这是高于此阈值的抗生素浓度, 临床断点。如何进行实验研究？如果少量的细菌种群受到高剂量抗生素的挑战, 就像在 Lenski 的实验中那样, 最可能的结果是抗生素会驱使所有的人群灭绝。同时, 如果使用的抗生素剂量低, 低于最低限度的抑制浓度 (MIC) 的父母菌株, 那么它是不可能的细菌群体将发展临床相关水平的抗药性, 特别是如果阻力承载着很大的成本。这两种情况的一个折中方案是使用 "进化营救" 实验9,10,11。在这种方法中, 大量的文化 (通常 > 40) 受到挑战, 随着时间的推移, 抗生素的剂量增加, 通常是通过加倍抗生素浓度每天12。这个实验的特点是, 任何没有进化出抗药性的种群都将被迫灭绝。以这种方式受到挑战的大多数人口将被驱赶绝种, 但少数族裔将坚持不断发展的高水平的抵抗。本文介绍了该实验设计如何能够用于研究多拷贝产质粒对抗性进化的贡献。
细菌通过两条主要途径、染色体突变和获取移动遗传元素, 主要是质粒13获得对抗生素的抗药性。质粒在抗生素耐药性的演变过程中起着关键作用, 因为它们能够通过共轭14,15在细菌间转移抗性基因。质粒可根据其大小和生物学分为两组: "小", 每个细菌细胞的拷贝数和 "大", 低拷贝数16,17。大型质粒在抗生素耐药性演变中的作用已被广泛记录在案, 因为它们包括共轭质粒, 这是细菌传播抗药性和多种抗药性的关键驱动因素15。在细菌17、18中, 小多拷贝产质粒也非常常见, 它们经常编码抗生素耐药性基因19。然而, 小多拷贝产质粒在抗生素耐药性演变中的作用在较小程度上被研究。
在最近的一项工作中, 我们建议多拷贝产质粒可以通过增加基因突变率来加速它们携带的基因的进化, 因为每个细胞的基因复制数都是12。使用具有大肠杆菌菌株 MG1655 和β-酶基因血乳酸TEM-1的实验模型, 表明多拷贝产质粒加速了 TEM-1 突变的出现率, 赋予了第三代抗性头孢他啶。结果表明, 多拷贝产质粒在抗生素耐药性的演变过程中可能起着重要的作用。
在这里, 我们提供了一个详细的描述, 我们已经开发的方法来研究多拷贝产质粒介导的抗生素耐药性的演变。该方法有三种不同的步骤: 一是在多拷贝产质粒或宿主细菌的染色体中插入研究中的基因。第二, 利用实验进化 (进化拯救) 来评估不同菌株的潜能, 以适应选择性压力。第三, 用 DNA 测序法确定质粒介导的进化的分子基础, 并在父母基因型中单独重建疑似突变。
最后, 虽然这里描述的协议是为了调查抗生素耐药性的演变而设计的, 但人们可以争辩说, 这种方法对于分析任何多拷贝产突变所获得的创新的进化是很有用的。质粒编码基因。

<table border="0" cellpadding="0" cellspacing="0" style="width:668px;" width="668">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thermocycler</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">C1000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electroporator</td>
			<td style="width:167px;">BiorRad</td>
			<td style="width:119px;">1652660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electroporation cuvettes</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z706078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NanoDrop 2000/2000c</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">ND-2000</td>
			<td>Determine DNA quality measuring the ratios of absorbance 260nm/280nm and 260nm/230nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubator</td>
			<td style="width:167px;">Memmert</td>
			<td style="width:119px;">UF1060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubator (shaker)</td>
			<td style="width:167px;">Cole-Parmer Ltd</td>
			<td style="width:119px;">SI500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrophoresis power supply</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">1645070</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrophoresis chamber</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">1704405</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pippettes</td>
			<td style="width:167px;">Biohit</td>
			<td style="width:119px;">725020, 725050, 725060, 725070</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Multi-channel pippetes</td>
			<td style="width:167px;">Biohit</td>
			<td style="width:119px;">728220, 728230, 
			728240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plate reader Synergy HTX</td>
			<td style="width:167px;">BioTek</td>
			<td style="width:119px;">BTS1LF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inoculating loops</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">I8388</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well plates</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:119px;">351172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB</td>
			<td style="width:167px;">BD Difco</td>
			<td style="width:119px;">DF0446-17-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB agar</td>
			<td style="width:167px;">Fisher scientific</td>
			<td style="width:119px;">BP1425-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phusion Polymerase</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">F533S</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:216px;">Gibson Assembly</td>
			<td style="width:167px;">New England Biolabs</td>
			<td style="width:119px;">E2611S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Resctriction enzymes</td>
			<td style="width:167px;">Fermentas FastDigest</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:216px;">Antibiotics</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:167px;">Qiagen</td>
			<td style="width:119px;">27104</td>
			<td>Plasmid extraction kit</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:216px;">Wizard Genomic DNA Purification Kit</td>
			<td style="width:167px;">Promega</td>
			<td style="width:119px;">A1120</td>
			<td>gDNA extraction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNeasy Blood &#38; Tissue Kits</td>
			<td style="width:167px;">Qiagen</td>
			<td style="width:119px;">69506</td>
			<td>gDNA extraction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electroporation cuvettes</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z706078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dishes</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryotubes</td>
			<td style="width:167px;">ClearLine</td>
			<td style="width:119px;">390701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well plates (-80&#186;C storage)</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">249945</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QuantiFluor dsDNA System</td>
			<td style="width:167px;">Promega</td>
			<td style="width:119px;">E2670</td>
			<td>Quantification of DNA concentartion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">1613100</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50x TAE buffer</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">1610743</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T4 Polynucleotide Kinase</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">EK0031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T4 DNA Ligase</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">EL0014</td>
			<td></td>
		</tr>
	</tbody>
</table>

testing the role of multicopy plasmids in the evolution of antibiotic resistance

多拷贝产质粒在原核生物中非常丰富, 但它们在细菌进化中的作用仍不甚了解。我们最近发现, 多拷贝产质粒提供的每个细胞基因拷贝数的增加, 都可以加速粒编码基因的进化。在这项工作中, 我们提出了一个实验系统, 以测试多拷贝产质粒的能力, 促进基因进化。使用简单的分子生物学方法, 我们构建了一个模型系统, 在这种模式下, 抗生素耐药性基因可以插入到大肠杆菌 MG1655, 无论是在染色体或多拷贝产质粒. 我们使用一种实验进化方法在越来越多的抗生素浓度下传播不同的菌株, 我们测量细菌种群的存活时间。抗生素分子和抗性基因的选择是为了使基因只能通过获取突变来获得抵抗。这种 "进化拯救" 方法提供了一种简单的方法来检测多拷贝产质粒的潜能, 以促进抗生素耐药性的获取。在实验系统的下一步, 对抗生素耐药性的分子基础进行了表征。为了找出负责获取抗生素耐药性的突变, 我们使用从整个种群和克隆获得的样本的深 DNA 测序。最后, 为了确认基因突变在研究中的作用, 我们在父母的背景下重建它们, 并测试结果菌株的抗性表型。

在我们以前的研究中, 研究了β-酶基因乳酸TEM-1对第三代头孢霉素12抗性的进化。这个基因之所以被选择是因为, 虽然 TEM-1 并没有对头孢他啶的抗性, 但在血乳酸TEM-1中的突变可以扩大 TEM-1 的活性范围, 以水解以甲霉素29为孢菌素抗生素耐药性酶的突变, 如β-lactamases 或氨基糖苷类改变酶导致其活动范围的变化是常见的29,30。该实验系统是研究这种酶的进化的理想方法。有关此协议之后成功实验的详细报告, 请参阅 San 文澜et。201612。
在这里, 给出了这个实验系统的可能结果的一个例子来说明协议 (请注意, 这个例子使用的数据不是真实的)。探讨多拷贝产质粒在抗生素耐药性基因进化中的潜在作用在这个例子中 (让我们称它为瑞莎), 我们开发了上文所述协议1节之后的实验系统。实验产生三株: MG1655, MG1655::瑞莎和 MG1655/pRESA。对两种不同的ß-内酰胺抗生素 (头孢他啶和美罗培南) 的耐药性的演变是根据该议定书2节所述步骤进行的。图 2显示了正在研究中的种群的生存曲线。在这个例子中, 与 MG1655 或 MG1655 的人相比, 头孢他啶 MG1655/pRESA 的种群存活显著增加::瑞莎(对数秩测试, P< 0.05)。另一方面, 在美罗培南的情况下, 不同品系的种群生存率没有显著差异 (对数秩试验, P> 0.05)。因此, 这些结果表明, 基因瑞莎在多拷贝产质粒中的存在增强对头孢他啶的抗性进化, 而不是美罗培南。
在实验的最后一步, 研究了抗生素耐药性的分子基础, 如《议定书》3节所解释的那样。首先, DNA 测序将揭示瑞莎中可能对抗性表型造成的突变。其次, 重建父母 MG1655 (染色体和质粒) 中的瑞莎突变将证实或放弃其对抗生素耐药性表型的作用。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57386/57386fig1.jpg"> 图 1.协议的不同阶段的示意图表示形式.从左到右: (一) 实验系统的构造: MG1655, MG1655::瑞莎和 MG1655/pRESA。细菌染色体表现为棕色, 质粒为蓝色,瑞莎基因为红色。(二) 进化抢救方法, 实验性地发展抗生素耐药性: 在抗生素浓度不断增加的情况下, 不同菌株的几个种群被传播。(三) 分析抗生素耐药性的分子基础: 从进化的种群和克隆中采集 DNA 样本, 检测抗生素耐药性突变, 以及重建父母株中的这些突变。<a href="/files/ftp_upload/57386/57386fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57386/57386fig2.jpg"> 图 2。生存曲线随着抗生素浓度的增加.表示属于菌株 MG1655、MG1655:瑞莎和 MG1655/pRESA 的可行种群数量。在抗生素头孢他啶和美罗培南浓度不断增加的情况下, 每株菌株的48个种群被传播, 开始于1天的1/8 的麦克风和每天加倍的抗生素浓度。红色虚线垂直线代表研究中抗生素的麦克风。请注意, 在头孢他啶的情况下, 随着时间的推移, 属于不同菌株的种群的存活率存在显著差异 (对数秩测试, P< 0.05)。关键的是, 只有携带该质粒的种群才能生存到高水平的抗生素浓度。另一方面, 在美罗培南的情况下, 不同种群的生存时间 (对数秩测试, P> 0.05) 没有显著差异。<a href="/files/ftp_upload/57386/57386fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance at JoVE.com

测试多拷贝质粒在抗生素耐药性进化中的作用|遗传学 |视频

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

多拷贝产质粒在抗生素耐药性演变中的作用试验

在此, 我们提出了一个实验方法来检验多拷贝产质粒在抗生素耐药性进化中的作用。

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

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11.5K 次观看.  Universidad Complutense de Madrid. 在此, 我们提出了一个实验方法来检验多拷贝产质粒在抗生素耐药性进化中的作用。

视频: Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

设计与低成本的使用，自动Morbidostat细菌的适应性进化在抗生素药物的选择

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

科研

遗传学

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

程序的微生物实验室自适应演化的恒化

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5&#39;-TA-3&#39; is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5&#39; end to a 4&#39;-hydroxymethyl-4,5&#39;,8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

工具来研究蛋白质建筑HMGB1的作用，螺旋窜改，站点特定的DNA间交联处理

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction.

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

用于抗倍体植入前基因测试的半导体测序

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of individual cell populations often makes it difficult to obtain enough material for molecular studies. For example, the accessory gland of the Drosophila male reproductive system contains two distinct secretory cell types. The main cells make up 96% of the secretory cells of the gland, while the secondary cells (SC) make up the remaining 4% of cells (about 80 cells per male). Although both cell types produce important components of the seminal fluid, only a few genes are known to be specific to the SCs. The rarity of SCs has, thus far, hindered transcriptomic analysis study of this important cell type. Here, a method is presented that allows for the purification of SCs for RNA extraction and sequencing. The protocol consists in first dissecting glands from flies expressing a SC-specific GFP reporter and then subjecting these glands to protease digestion and mechanical dissociation to obtain individual cells. Following these steps, individual, living, GFP-marked cells are sorted using a fluorescent activated cell sorter (FACS) for RNA purification. This procedure yields SC-specific RNAs from ~40 males per condition for downstream RT-qPCR and/or RNA sequencing in the course of one day. The rapidity and simplicity of the procedure allows for the transcriptomes of many different flies, from different genotypes or environmental conditions, to be determined in a short period of time.

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

Here, we present a protocol to dissociate and sort a specific cell population from the Drosophila male accessory glands (secondary cells) for RNA sequencing and RT-qPCR. Cell isolation is accomplished through FACS purification of GFP-expressing secondary cells after a multistep-dissociation process requiring dissection, proteases digestion and mechanical dispersion.

从果蝇雄性附件腺的二级细胞中分离RNA的FACS协议

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of ...

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

Our experimental approach provides a strategy to follow plasmid abundance and antibiotic resistance over time in bacterial populations.

实验进化方法中疟原虫介导抗生素耐药性的定量化

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Presented here is a stepwise procedure for in vitro differentiation of human primary keratinocytes by contact inhibition followed by characterization at the molecular level by RNA-seq analysis.

通过RNA-Seq分析，人类原主角细胞的体外分化特征

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...