本研究得到了国家自然科学基金（U20A20118）和生态环境研究所湖南省洞庭湖防灾减灾技术监测与防灾减灾技术湖南省工程研究中心开放基金（2023-DTH-04）的支持。

所有动物实验均经中国科学院亚热带农业研究所伦理委员会批准。
1.防啮齿动物墙的建造
<ol><li>挡浪墙的施工<ol><li>建造比路堤面高0.5米的挡浪墙。用水泥将湖边的墙面平整，并在顶部添加一块比墙稍宽的平板，使其延伸 8 厘米，类似于舌头（图 1）。这确保了啮齿动物无法爬墙。 注：挡浪墙的高度和平板的宽度可根据不同的啮齿动物进行建造。</li>
	</ol></li>
	<li>挖防啮齿动物的沟渠<ol><li>当一些挡浪墙有通向海滩的缝隙时，在缝隙处挖一条防啮齿动物的沟渠。沟渠深0.5米，比挡浪墙的缝隙略宽。</li>
		<li>在沟渠中填土或覆盖薄水泥板，以方便行人和车辆通过。在汛期，去除薄水泥板并清除沟渠中的土壤，从而完全阻断啮齿动物的迁徙路径（图2）。</li>
	</ol></li>
	<li>埋盆屏障法<ol><li>首先，沿着堤坝竖起围栏。</li>
		<li>由纤维板、塑料薄膜和木板组成挡板，并用木桩支撑。</li>
		<li>将板栅栏埋入土壤5-10厘米，高度为0.5 m。将深盆以 50 m 的间隔埋在固定围栏之间。</li>
		<li>确保花盆深 80 厘米，直径 60 厘米，并埋在挡板旁边的土壤中，花盆口与地面齐平（图 3）。</li>
		<li>啮齿动物接近堤坝，沿着栅栏行走，然后被引导到花盆中。最后，疏通花盆并清理啮齿动物。</li>
	</ol></li>
</ol>2. 效率统计方法
<ol><li>捕捉陷阱法<ol><li>在整个调查期间使用捕捉器，使用生葵花籽作为诱饵。沿线样带对三到四个地块进行采样，每个地块约 6-10 公顷。确保地块之间的距离为 >150 m，并在每个地块中安装 80-100 个陷阱，每 5 m 放置一个陷阱。</li>
		<li>每年春末夏初水位上升后，对相应调查湖的农田进行调查。</li>
		<li>沿田脊以5米的间隔铺设陷阱，每个区域放置>200个陷阱。下午放置陷阱，第二天早上收集。然后，根据物种计算捕获的啮齿动物。</li>
		<li>使用以下公式计算相对丰度，这是陷阱成功的指示： <img alt="Equation 1" src="/files/ftp_upload/66596/66596eq01.jpg" style="margin: auto;"> 其中 A 是捕获的啮齿动物数量， B 是陷阱的有效数量， C 是啮齿动物的相对丰度。</li>
	</ol></li>
	<li>农作物损失统计<ol><li>如果农作物位于湖滩堤坝的另一侧，则使用农作物损害统计数据来控制啮齿动物的影响16.<ol><li>在进行啮齿动物相对丰度调查的同时，对调查区域内的稻田进行随机抽样。</li>
			<li>根据单一的五点对角线采样方法对所选字段进行采样，从每个字段收集五个样本方块。在每个方形样品中收集水稻幼苗（10×10株植物）。</li>
		</ol></li>
		<li>记录幼苗总数和被啮齿动物折断的幼苗数量，以确定损坏率。通过计算啮齿动物在控制啮齿动物之前和之后因损害而产生的成本来确定啮齿动物控制的有效性。</li>
	</ol></li>
	<li>诱饵消耗方式<ol><li>在调查区域放置相同大小的诱饵，一定时间后，计算诱饵消耗率作为啮齿动物密度的指标17。<ol><li>如果使用的诱饵是大米，则以重量（g）计算诱饵消耗量。同时，控制诱饵中水分的自然减少，以纠正诱饵消耗。如果诱饵是大颗粒或块状物，例如红薯，请观察并记录消耗的颗粒或块状物的数量。</li>
		</ol></li>
	</ol></li>
	<li>开孔法<ol><li>堵住原来的孔洞，减少对废弃孔的误判。</li>
		<li>通过比较啮齿动物在农田中挖的洞的数量，确定有或没有防啮齿动物墙的啮齿动物预防的有效性。 注意：堵塞孔应严格进行，最好在大鼠活动高峰之前进行，例如晚上的家鼠或清晨的地松鼠。</li>
	</ol></li>
</ol>3. 统计分析
<ol><li>使用适当的数据分析软件分析数据，并使用卡方检验 （χ2） 确定每个捕获率的显著性，统计显著性设置为 P <0.05。 注：SPSS用于分析数据。其他统计软件也可用于统计分析，如DPS。</li>
</ol>

通过防啮齿动物结构减少农业中的啮齿动物侵扰

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作者没有什么可透露的。

该协议中有几个关键步骤。防啮齿动物墙舌形平板的宽度应足够宽，并且应将墙的高度设置为该区域的啮齿动物无法越过它。当挡浪墙有通向海滩的缝隙时，防啮齿动物沟槽的深度应设置为啮齿动物无法攀爬的深度，沟渠的宽度应设置为啮齿动物无法跨越的距离。埋盆屏障法中挡板和花盆的高度应设置为啮齿动物无法跨越的高度。当一个地区发生啮齿动物爆发时，应定期清理花盆中的啮齿动物，?...

啮齿动物的危害是一种重要的生物灾害，对人类生产和生活的各个方面造成广泛的破坏1,2。在农业中，农田的啮齿动物侵扰会损害农作物3;在林业中，啮齿动物以树苗、树根、树皮和植物种子为食，导致森林更新延迟和树木死亡，进而影响森林绿化和固沙4;在草原上，啮齿动物以根和种子为食，导致草原植被退化和砂土增加，影响草原畜牧业的发展5.此外，啮齿动物是许多病毒、细菌和寄生虫的宿主，可严重危害人类健康6.
洞庭湖位于湖南省东北部，是我国重要的蓄水抑洪湖7。它具有许多生态功能，如防洪和滞留、生物多样性保护和水资源供应8,9。近几十年来，洞庭湖地区发生过多次啮齿动物暴发，特别是2007年芦苇田鼠Microtus fortis的暴发，造成了巨大的经济损失10。在旱季，M. fortis 在洞庭湖地区的湖滩上生长和繁殖。夏季汛期，洞庭湖水位上升，汛期，桫椤的栖息地不断缩小，迫使其通过游泳、越过防洪堤、到达附近的农田等方式迁入堤坝，对农业生产造成极大危害11,12。Chen等人基于位于湖南省益阳市的金边农场和南大镇共同创建的防浪挡土墙方法，提出了一种建造防啮齿墙阻断鲈桐迁徙路径的控制措施13,14。在没有防啮齿动物墙的堤坝中，大量的 M. fortis 会在迁徙期间和迁徙后对农作物造成毁灭性的破坏。没有防啮齿动物墙的堤坝通常是人工捕获和下药，以在啮齿动物进入农田期间和之后消灭它们。在具有防啮齿动物墙的堤坝的情况下，许多啮齿动物在 M. fortis 爆发期间仍留在堤坝外。因此，许多诱捕和消除作业是在堤坝外进行的;一般来说，农田不需要实施化学药物或人工诱捕。这种方法可以显着降低害虫的密度，而不会对农田作物造成严重伤害。常规的啮齿动物药物预防方法无法达到预期效果，存在与环境污染和人畜安全相关的隐患15。考虑到啮齿动物容易爆发并造成灾害的特点，以下方案介绍了三种物理防鼠方法，包括永久性防啮齿动物墙和两种临时墙，并提出了四种测量啮齿动物控制效果的统计方法，为啮齿动物控制提供了科学依据。代替传统的毒饵，防啮齿墙有效保护环境，保障人类和动物健康;因此，它是一种更有效、更环保的控制方法，应该被提倡和大力推广。

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	<tbody>
		<tr>
			<td>Snap traps</td>
			<td>Guixi Mousing Tool Factory, Jiangxi, China</td>
			<td>large-sized</td>
			<td>150 mm &#215; 80 mm</td>
		</tr>
		<tr>
			<td>SPSS</td>
			<td>IBM</td>
			<td>version 16.0&#160;</td>
			<td></td>
		</tr>
	</tbody>
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rodent-proof wall: an efficient physical method for controlling rodents and its efficiency statistics

啮齿动物的危害对农作物、人类生命和健康构成重大威胁。与化学防治啮齿动物（例如放置有毒诱饵）相比，使用物理方法（例如建造防啮齿动物墙）更经济、更环保。本研究介绍了一种物理控制有害啮齿动物的方法和四种计算啮齿动物控制效果的方法。为了解防鼠墙的控制效果，于2012年4月和7月对洞庭湖滩和堤防内的相应农田进行了调查。结果表明，人工诱捕和药物灭药后，有防鼠墙的农田芦苇 田鼠密度 为0.52%，显著低于无防鼠墙的农田（1.76%）（χ2 = 3.900，P = 0.048）。 有防啮齿动物墙的堤坝迁移到农田的 松 树密度下降了98.53%，显著高于无防鼠墙的堤坝密度下降的86.61%（χ2 = 11.060，P = 0.01）。 结果证明了防啮齿动物墙控制的有效性。因此，应倡导并大力推动建造防啮齿动物墙，防止啮齿动物迁徙到洞庭湖地区和类似环境中，因为它们会造成危害。

为确定防啮齿动物墙的控制效果，分别于2012年4月和7月对湖滩和相应的堤坝农田进行了水位上升前后的调查15。勘察地点位于湖南省洞庭湖区，即岳阳县马塘源外湖滩（北纬29°14.5′;东经113°03.2′）、大同湖区北洲子镇（北纬29°10.1′;东经112°47.7′）、元江市南大镇双丰堤（北纬29°1.3′;东经112°45.2′）和元江市创业园南嘴镇木南村堤外牟平湖（北纬28°59.6′;东经112°15.1′）。北...

Watch this Scientific Journal Video about Author Spotlight: Impact of Physical Barriers on Rodent Populations in Farmland Areas at JoVE.com

作者聚焦：物理屏障对农田地区啮齿动物种群的影响

在这里，我们介绍了三种对害虫啮齿动物进行物理防治的方法，四种计算它们对啮齿动物有效性的方法，以及建造防啮齿动物墙的效果统计。

防啮齿动物墙：一种有效的控制啮齿动物的物理方法及其效率统计

Rodent damage poses a significant threat to crops, human life, and health. Compared to chemical rodent control, such as placing poisonous baits, it is ...

啮齿动物的危害对农作物、人类生命和地球构成重大威胁。本研究介绍了一种物理方法，即防啮齿动物墙，该方法适用于防止洞庭湖地区具有迁徙习性的啮齿动物，如Microtus fortis。他们从湖滩迁移到农田，这造成了伤害。啮齿动物对湖滩内的生态系统有益，但当它们向农田迁移时却是有害的。很难找到一种方法来控制中心区域的啮齿动物而不对其他区域造成伤害。在洞庭湖地区，防啮齿动物墙可能是一个好主意。建造防啮齿动物的墙可以提供长期保护。这意味着将啮齿动物控制在一定区域内，而不是直接杀死它们。这对生态系统和环境是友好的。在洞庭湖地区，防鼠墙由原来的挡浪墙改造而成，成本低廉。

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Biology

Rodent-Proof Wall: An Efficient Physical Method for Controlling Rodents and its Efficiency Statistics

500 次观看.  Chinese Academy of Sciences. 啮齿动物的危害对农作物、人类生命和地球构成重大威胁。本研究介绍了一种物理方法，即防啮齿动物墙，该方法适用于防止洞庭湖地区具有迁徙习性的啮齿动物，如Microtus fortis。他们从湖滩迁移到农田，这造成了伤害。啮齿动物对湖滩内的生态系统有益，但当它们向农田迁移时却是有害的。很难找到一种方法来控制中心区域的啮齿动物而不对其他区域造成伤害。在洞?...

视频: 作者聚焦：物理屏障对农田地区啮齿动物种群的影响

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

传统美国宇航局后肢卸小鼠模型的一个交替的方法

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

科研

生物学

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

对于外来体定量和尺寸测量新方法研究

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

组织化学染色<em&gt;拟南芥</em&gt;次生壁元素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.

DNA的亲和纯化的芯片（DAP芯片）方法来确定目标基因的细菌双组分调控系统

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of ...

An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett&#8217;s esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.

An Immunofluorescent Method for Characterization of Barrett&amp;#8217;s Esophagus Cells

There is a discernible need for improved information on molecular drivers of Barrett&#8217;s Esophagus. Immunofluorescent staining is a useful technique for understanding the effects of cell signaling on cell morphology. We present a simple, effective protocol for the use of immunofluorescent staining to assess therapeutic treatment in Barrett&#8217;s Esophagus cells.

免疫荧光法Barrett食管细胞的表征

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

三维球体制作一个高效，灵活细胞聚集方法

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

一种有效的体外转位方法由转录调节的睡眠美容系统封装到一个集成缺陷的慢向量

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

一种改进的小鼠原代肝细胞分离时间和劳动效率方案

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...