1) 셀 준비 <ol><li> 자기편 세포를 사용하는 경우, 그것은 trypsinize과 electroporation하기 전에 세포를 수집하는 것이 필요합니다. </li><li> 세 가지 통로 번호를 나타내는 네 개의 마우스 배아 fibroblast (MEF) 문화 간의 transfection을 비교하려면 각각의 플라스크에서 다음을 수행합니다. </li><li> 세포 배양 매체를 대기음. </li><li> 세포를 씻어 PBS를 추가합니다. </li><li> PBS를 제거, 세포를 커버하는 충분한 트립신을 추가하고, 트립신은 세포를 분리 수 있도록 몇 분 기다리십시오. </li><li> , 세포의 상태를 확인하기 위해 현미경으로 flasks을 확인 세포를 분리 플라스크 갈기하고 있는지 세포가 모두 떨어뜨려하기 위해 다시 플라스크를 확인합니다. </li><li> 추가 시간과 반복 필요한 경우 기다립니다. </li><li> 모든 세포가 분리되면 트립신를 무력화하기 위해 혈청이 포함된 미디어를 추가할 수 있습니다. </li><li> 원심 분리기 튜브에 세포를 전송하고, 원심 분리에 의해 펠릿 세포 (RCF = 300 XG). </li><li> PBS의 유명한 볼륨에 뜨는 및 resuspend 세포를 제거합니다. </li><li> 세포를 세어보세요. </li><li> 세포 현탁액의 해당 볼륨이 실험을 위해 세포 (당신은 1 X 10 6 세포 / ML의 밀도에 잘 당 세포 150 μL가 필요합니다)의 필요한 숫자를 제공하는 새로운 튜브로 전송합니다. </li><li> 원심 분리기 세포. </li><li> 1 X 10 6 세포 / ML의 세포 밀도를 달성하기 위해 진 Pulser의 electroporation 버퍼의 해당 볼륨에 뜨는 및 resuspend 세포를 제거합니다. </li><li> 세포 현탁액의 플라스미드 당 ML 20 μg을 추가하고 부드럽게 섞는다. </li></ol> 2) Electroporation 선박 설정 및 electroporation 플레이트 설치 및 electroporation <ol><li> 진 Pulser MXcell의 electroporation 시스템의 전원 모듈에 플러그 플레이트 챔버. </li><li> 피펫 150 μL 세포 혼합물 또는 96 - 웰 electroporation 판의 우물에 버퍼. </li><li> 플레이트 챔버 및 펄스의 접시를 넣어. </li><li> 챔버에서 플레이트를 제거합니다. </li><li> 각 우물에 위아래로 pipetting으로 잘 내용물을 섞는다. </li><li> 각 우물에서 12 잘 접시에 미리 예열 버퍼 세포를 전송합니다. </li><li> 세포를 배포하고 배양기에 넣어 접시 누릅니다. </li><li> 세포는 24 시간 동안 회복하자. </li></ol> 쿠베트 설정 및 electroporation <ol start="9"><li> ShockPod의 유전자 Pulser MXcell의 electroporation 시스템 및 플러그 ™ 쿠베트 챔버의 전원 모듈에서 플레이트 챔버를 뽑습니다. </li><li> 0.4 cm 갑 electroporation의 쿠베트에 세포 현탁액의 피펫 600 μL. </li><li> ShockPod 챔버에 쿠베트를 넣고 전기 펄스를 제공합니다. </li><li> 챔버에서 쿠베트를 제거합니다. </li><li> 쿠베트에 아래 pipetting하여 쿠베트 내용을 섞는다. </li><li> 각 우물에서 12 잘 접시에 미리 예열 버퍼 세포를 전송합니다. </li><li> 세포를 배포하고 배양기에 넣어 접시 누릅니다. </li><li> 세포가 24 시간 동안 회복하자. </li></ol> 3) 대표 결과 세포를 transfecting하고 복구할 수있게되면, 유동세포계측법를 사용하여 양적 epifluorescent 현미경을 사용하여, 질적으로 transfection 효율을 분석합니다. <img src="/files/ftp_upload/1662/1662fig1.jpg" alt="그림 1" /> 성공적으로 electroporated되었습니다 지금은 GFP 유전자를 표현 아르 그림 1. 세포는 epifluorescent 현미경 아래에 나타납니다. <img src="/files/ftp_upload/1662/1662fig2.jpg" alt="그림 2" /> 그림 2. 위상 대조에서 세포를보기는 transfected와 untransfected 모두 세포의 시각화 수 있습니다. 이들은 200V의 낮은 전압 electroporation 펄스에 노출되었던 세포 수 있습니다. 세포 인해 높은 세포 밀도에 크게 합류하고 있습니다. <img src="/files/ftp_upload/1662/1662fig3.jpg" alt="그림 3" /> 그림 3. epifluorescence 아래보기의 동일한 필드는 세포가 GFP 마커를 표현 아르의 숫자를 보여주지만, 이들은 이전의 이미지에서 보이는 세포의 단지 작은 비율입니다. <img src="/files/ftp_upload/1662/1662fig4.jpg" alt="그림 4" /> 그림 4. 250V에서 위상 대조에 따라 본 사는 세포의 총 개수가 약간 줄어 듭니다. <img src="/files/ftp_upload/1662/1662fig5.jpg" alt="그림 5" /> epifluorescence에서 하나는 GFP 표현 세포의 수가 증가했다는 그림 5.보실 수 있습니다. <img src="/files/ftp_upload/1662/1662fig6.jpg" alt="그림 6" /> 그림 6. 가장 높은 전압에서이 375V를 적용, 눈에 보이는 적은 라이브 세포가 없습니다. <img src="/files/ftp_upload/1662/1662fig7.jpg" alt="그림 7" /> 그림 7. 그러나 남아있는 세포의 큰 비율 GFP를 표현하고 있습니다. 최적의 어떤 조건 것은 실험 설계에 따라 달라집니다. 어떤 실험에서 transfected 세포의 가장 큰 숫자는 다른 실험에서 가장 높은 비율 transfection이 최선이 될, 최적의 수 있습니다. 우리는 각각의 조건에 따라 긍정적인 GFP 방법과 비율이 세포 나이마다 다릅니다 세포의 비율에 관심이 있습니다. 유동세포계측법는 다른 electroporation 조건의 각에서 transfection 결과에 대한 정량적 정보를 제공할 수 있습니다. <img src="/files/ftp_upload/1662/1662fig8.jpg" alt="그림 8" /> 그림 8. 여기 12 electroporation 조건의 각 이하 5 세포가 게재되는 통로에 긍정적인 GFP 아르 세포의 비율입니다. 최대 transfection 비율이 가장 강한 정사각형 웨이브 펄스에서 높은 전압 지수 붕괴 펄스, 상태 6, 70 % 이하 약 80 %입니다 상태 12, 테스트. <img src="/files/ftp_upload/1662/1662fig9.jpg" alt="그림 9" /> 그림 9. 세포 전에 electroporation 9 회 합격으로 transfection 비율의 전반적인 패턴이 거의 동일하지만, transfection 비율에서 아주 약간의 감소와 함께. <img src="/files/ftp_upload/1662/1662fig10.jpg" alt="그림 10" /> 그림 10. 여기에 표시가 통로 젊은 세포에 비해 transfection 비율에 표시된 감소를 보여 13 세포에 GFP 세포의 비율입니다. 가장 높은 transfection 비율은 최대한 빨리 격리 후 건강한 세포를 사용하는의 중요성을 보여주는 젊은 세포로 달성했던 약 절반되었습니다. <table border="1"><tbody><tr><td colspan="3"> 진 Pulser MXcell를 사용하여 transfecting MEF 세포에 사용 Electroporation 조건 </td></tr><tr><td> 조건 (1-6) 지수 붕괴 펄스, 350 UF, 1000ohm 모든 </td><td> 전압 (V) </td><td></td></tr><tr><td> 1 </td><td> 200 </td><td></td></tr><tr><td> 2 </td><td> 250 </td><td></td></tr><tr><td> 3 </td><td> 300 </td><td></td></tr><tr><td> 4 </td><td> 326 </td><td></td></tr><tr><td> 5 </td><td> 350 </td><td></td></tr><tr><td> 6 </td><td> 376 </td><td></td></tr><tr><td> 조건 (7-12) 스퀘어 웨이브 펄스, 2,000 UF, 1000 옴, 1 펄스 모든 </td><td> 전압 (V) </td><td> 펄스 기간 (MS) </td></tr><tr><td> 7 </td><td> 200 </td><td> 10 </td></tr><tr><td> 8 </td><td> 250 </td><td> 10 </td></tr><tr><td> 9 </td><td> 300 </td><td> 10 </td></tr><tr><td> 10 </td><td> 200 </td><td> 20 </td></tr><tr><td> 11 </td><td> 250 </td><td> 20 </td></tr><tr><td> 12 </td><td> 300 </td><td> 20 </td></tr></tbody></table>

저자는이 문서에서 사용되는 시약 및 장비를 생산하는 바이오 래드 연구소에 의해 고용되어

이 동영상이 문서에서는 쉽게 MEFs 또는 기타 기본 세포 라인에 대한 최적의 electroporation 조건을 식별하는 MXcell의 electroporation 시스템을 사용하는 방법을 보여줍니다. 많은 사람들이 많은 별도의 실험에 대한 필요성을 없앨 수 동시에 수행하는 실험이나 최적화 조건의 복제에 대한 96 - 웰 플레이트 형식 있습니다. 이 절차를 수행하는 동안, 하나는 가능한 한 빨리 격리 후 건강한 세포를 사용하고 electroporation 버퍼에 일치 electroporation 조건을 사용하는 기억한다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Gene Pulser&reg; Electroporation Buffer</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2676</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser MXcell&trade; Electroporation System</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2670</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser MXcell&trade; ShockPod&trade; Cuvette Chamber</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2673</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser MXcell&trade; Electroporation System With ShockPod&trade; Cuvette Chamber</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2674</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

using the gene pulser mxcell electroporation system to transfect primary cells with high efficiency

그것은 electroporation은 기본 세포에 플라스미드 DNA 또는 siRNA을 소개하는 가장 효과적인 방법임을 더욱 분명 해지고 있습니다. 진 Pulser MXcell의 electroporation 시스템과 진 Pulser의 electroporation 버퍼 (바이오 래드)은 특히 포유 동물 세포를 쉽게와 같은 기본 및 줄기 세포와 같은 어려운 transfect 세포로 핵산을 transfect하기 위해 개발되었습니다. 우리는 신속하게 최고의 electroporation 조건을 식별하는 간단한 실험을 수행하는 방법을 보여줍니다 것입니다. 우리는 실험이 최적화가 수행 동시에 실시 할 수 있도록 electroporation 조건의 범위를 통해 몇 가지 샘플을 실행하는 방법을 보여줍니다 것입니다. 우리는 또한 같은 electroporation 효율을 유지하면서 최적의 조건, 표준 electroporation의 cuvettes와 함께 사용할 수 있습니다 96 - 웰 플레이트 electroporation을 사용하여 electroporation의 cuvettes에 electroporation 플레이트의 스위치를 촉진 식별 방법을 보여줍니다. 동영상에서 우리는 electroporation 실험의 성공 또는 실패를 가져올 수있는 핵심 요소 중 일부를 설명합니다.

Watch this Scientific Journal Video about Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency at JoVE.com

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

이 절차를 빠르게 쉽게 마우스 배아 섬유아 세포 (MEFs) 또는 기타 기본 세포에 가장 적합한 electroporation 조건을 식별하는 유전자 Pulser MXcell의 electroporation 시스템을 사용하는 방법을 보여줍니다. 문제 해결을위한 고려 사항도 관련 비디오 설명합니다.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

using-gene-pulser-mxcell-electroporation-system-to-transfect-primary

Research

JoVE Journal

Biology

23.9K 조회수.  Bio-Rad Laboratories, Inc.. 이 절차를 빠르게 쉽게 마우스 배아 섬유아 세포 (MEFs) 또는 기타 기본 세포에 가장 적합한 electroporation 조건을 식별하는 유전자 Pulser MXcell의 electroporation 시스템을 사용하는 방법을 보여줍니다. 문제 해결을위한 고려 사항도 관련 비디오 설명합니다.

비디오: Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

연구

생물학

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

마우스 망막 신경절 세포의 유전자 발현에 대한 utero 및 전직 생체내의 Electroporation에

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

차 문화와 Corti의 Murine 오르간의 플라스미드 Electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

히타치 F - 7000 형광 분광 및 PicoGreen 염료를 사용하여 dsDNA의 부량

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

배양 된 포유 동물 세포의 생물 학적 경로의 유비퀴틴과 유비​​퀴틴 같은 시스템 레귤레이터를 확인하는 siRNA를 상영

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

Zebrafish의 유충의 여러 어려운 대상 조직을 형질 심장 심실 사출을 사용하여 유전 모르 폴리 노의 접근을 반전

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

유동 세포 계측법에 의한 고효율 심근에 인간 다 능성 줄기 세포의 분화 및 특성

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

루시퍼 라제 리포터 유전자 RAW264.7 세포를 형질

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

유해 및 잠재적으로 유해한 구성 성분의 독성 효과를 평가하기 위해 높은 콘텐츠 심사 분석 (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...