이 작품은 슬로베니아어 연구기구 (프로젝트 J2 - 9770, infrastructural 센터 IP - 0510 및 프로그램 P2 - 0249)에 의해 지원되었다. 이 비디오는 &quot;Electroporation 기반 기술 및 트리 트먼트&quot;류블랴나, 슬로베니아 대학 전기 공학부 주최 과학 워크샵 및 대학원 과정에 대한 보충 자료를 나타냅니다. 저자는 친절 플라스미드 DNA를 제공하고 또한 Duša 홋직 감사합니다.

1. 세포 배양, 플라스미드 및 실험을위한 버퍼 준비 <ol><li> 본 실험 중국 햄스터 난소 세포는 (CHO - K1) 사용됩니다. 셀 2 MM L - 글루타민 10 % 태아 소 혈청, 400 μl / L gentamicin (시그마 - 알드리치 케미 GmbH를, Deisenhofen, 독일에서), 그리고 1 보충 영양 혼합 햄 - F12 (PAA)에 재배 ML / 리터 crystacilin (Pliva, 자그레브, 크로아티아). 전지는 37 24에 대한 보육의 humidified 5 % CO 2 분위기 ° C에 보관됩니다. </li><li> 확대 플라스미드 pEGFP - N1 (Clontech Laboratories의 주식, 마운틴 뷰, CA, 미국) 대장균 DH5α의 변형에 녹색 형광 단백질 (GFP)을 인코딩 및 HiSpeed​​ 플라스미드 맥시 키트 (Qiagen, 힐든, 독일)로 분리. 플라스미드 DNA의 농도는 (TE 버퍼에 용해 플라스미드) spectrophotometrically 260 nm의에서 결정하여 겔 전기 영동으로 확인한다. </li><li> isoosmolar 나트륨 인산 버퍼 (10 MM 나 2 HPO 4, 10 MM 아니 2 PO 4, 1 ㎜ MgCl 2, 250 MM의 자당, 산도 7.4) 준비합니다. </li><li> 실험 당일 0.25 % 트립신 / EDTA (에틸렌 다이아 민 테트라 초산) 솔루션 (시그마 - 알드리치 케미 GmbH를, Deisenhofen, 독일)과 trypsinization하여 세포 현탁액을 준비합니다. 4 1,000 RPM (180 XG)에서 5 분 원심 분리기 세포 ° C (시그마, 독일)와 5 세포 밀도 × 10 6 세포 / ML에 isoosmolar 인산 나트륨 버퍼에 resuspend 세포 펠릿. </li></ol> 2. 하드웨어 장비 <ol><li> 전지는 높은 전압 프로토 타입 발전기에 연결된 통합 전극과 피펫 팁의 전기장 (그림 1)에 노출되어 있습니다. 팁 및 전극 기하학은 비교적 균일한 전기장의 응용 프로그램을 허용하고 발전기가 다른 방향으로 전기 펄스의 전달을 허용합니다. 팁 및 발전기는 Biocybernetics의 연구소, 전기 공학부, 류블랴나 1 대학에서 개발되었다. </li></ol><img src="/files/ftp_upload/3309/3309fig1.jpg" alt="그림 1"/> 그림 1. 수직 및 수평 (A) 단면 및 사진 (b)는 통합 전극과 피펫 팁의. 단면 회색 색상의 전극에 대한 플라스틱 하우징 및 검정에 사용됩니다. 통합 전극과 피펫 팁 네 원통형 막대 전극으로 구성되어 있습니다. 전극은 스테인리스 스틸로 만들어진, 자신의 직경은 인접한 전극의 간격 1mm이며, 반대 전극의 간격 2mm 있으며, 1.4 mm이다. 전극은 병렬로 플라스틱 끝에 접착하고 해당 길이는 30mm 2입니다. 3. 진 electrotransfer 프로토콜 <ol><li> 농도 10 μg / ML에 세포 현탁액에 플라스미드 pEGFP - N1을 추가합니다. </li><li> 전기 펄스를 적용하기 전에, 상온에서 2~3분에 대한 혼합물을 품어. </li><li> 통합 전극과 피펫 팁에 세포 현탁액의 바램이 100 μl. </li><li> 최고의 유전자 electrotransfer의 효율을 달성하고 세포 생존을 유지하기 위해 전기 펄스의 최적의 매개 변수를 사용해야합니다. 이 실험에 8 직사각형 펄스 (각 1 Hz에서 반복 주파수 1 MS, 진폭 225 V의 기간과)의 기차는 고전압 프로토 타입 생성기를 사용하여 각 샘플에 적용됩니다. 두 개의 서로 다른 전기장 프로토콜은 (그림 2) 사용 : 두 번째 프로토콜 펄스에서 또는 전기장의 방향과 방향을 변경하여 전달됩니다 반면 첫 번째 프로토콜의 모든 펄스는 같은 방향으로 전달됩니다. 두 번째 프로토콜은 서로 다른 방향으로 전기 펄스의 응용 프로그램을 허용 적절한 펄스 발생기와 함께 사용할 수 있습니다. </li><li> 펄스 응용 프로그램은 6 자 플레이트에 피펫 팁의 세포를 전송 및 태아 송아지 혈청 (FCS - 시그마, 미국) (샘플 볼륨의 25 %) 추가 직후. </li><li> 37 5 분 세포를 품어 ° C 세포막의 resealing 수 있습니다. </li><li> 물론 6 각 샘플에 햄 - F12 2 ML를 추가하고 인큐베이터에 humidified 5 % CO 2 분위기에서 37 24 시간을위한 세포 ° C를 품어. </li></ol><img src="/files/ftp_upload/3309/3309fig2.jpg" alt="그림 2"/> . 그림 2 전기장 프로토콜 : (A) 모든 펄스가 같은 방향으로 전달됩니다은 (B) 펄스가 또는 전기장의 방향과 방향을 변경하여 전달됩니다. 4. 이미지 수집 및 유전자 electrotransfer의 효능의 결정 <ol><li> 유전자 electrotransfer의 효능은 펄스 신청 후 GFP 24 시간을 표현 세포의 비율로 결​​정됩니다. </li><li> 세포는 단색 시스템 (색채 IV, Visitron, 독일)와 배출이 507 nm의에서 감지로 생성된 488 NM에서 여기 광과 (우리의 경우에는 자이스 혈구 200 Axiovert, ZR 독일) 형광 현미경을 사용하여 관찰하고 있습니다. 이미지는 이미징 시스템 (MetaMorph 이미징 시스템, Visitr을 사용하여 기록됩니다에, 독일),하지만, 다른 유사한 수집 소프트웨어도 사용할 수 있습니다. </li><li> 20x 객관적인 확대에 적어도 다섯 이미지 (위상 콘트라스트와 녹색 형광) 취득. </li><li> 위상 콘트라스트 이미지와 녹색 형광 이미지에서 GFP를 표현 아르 세포에서 세포를 계산합니다. 각각의 해당 이미지의 모든 세포의 수를 (그림 3)과 GFP를 표현 아르 세포의 수를 나누어 유전자 electrotransfer의 효험의 백분율을 결정합니다. </li></ol> 5. 대표 결과 : <img src="/files/ftp_upload/3309/3309fig3.jpg" alt="그림 3"/> 그림 3. 모든 펄스가 같은 방향으로 전달되며 펄스가 또는 전기장의 방향을 변경하고 방향을 제시하여 전달됩니다되었을 때 GFP를 표현하는 세포의 비율입니다. 전지는 진폭 225 V, 기간 1 MS 1 Hz의 반복 주파수의 8 펄스의 기차에 노출되었습니다. 결과는 형광 현미경에 의해 취득되었다. 그래프의 각 값은 세 가지 독립적인 실험 ± 표준 편차의 의미를 나타냅니다. 전기장의 방향과 방향 GFP 증가를 표현 세포의 비율을 변경하여.

<ol>
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관심 없음 충돌 선언하지 않습니다.

진 electrotransfer 짧은, 고전압 전기 펄스 3 적용을 통해 세포로 DNA의 전송을 가능하게하고 안전성, 효능 및 응용 프로그램의 용이성으로 인해 바이러스 벡터에 대한 안전한 대안을 대표하는 다양한 생명 공학 기술입니다. 오늘날 유전자 electrotransfer 널리 4보고되었습니다이 방법을 사용하여 셀 및 첫 단계의 모든 종류의 I 임상 시험을 transfect하는 데 사용되지만, 기본 메커니즘은 아?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 햄 - F12 </td><td> PAA </td><td> E15 - 016 </td><td> 문화 매체 </td></tr><tr><td> L - 글루타민 </td><td> 시그마 - 알드리치 </td><td> G7513 </td><td></td></tr><tr><td> 태아 소 혈청 </td><td> PAA </td><td> A15 - 151 </td><td></td></tr><tr><td> gentamicin </td><td> 시그마 - 알드리치 </td><td> G1397 </td><td> 항생 물질 </td></tr><tr><td> crystacilin </td><td> Pliva </td><td> 625110 </td><td> 항생 물질 </td></tr><tr><td> pEGFP - N1 </td><td> Clontech Laboratories의 </td><td> 6085-1 </td><td> 플라스미드 DNA </td></tr><tr><td> HiSpeed​​ 플라스미드 맥시 키트 </td><td> Qiagen </td><td> 12,662 </td><td></td></tr><tr><td> 나 2 HPO 4 </td><td> 머크 </td><td> F640786 933 </td><td></td></tr><tr><td> 아니 2 PO 4 </td><td> TKI Hrastnik </td><td> 0795 </td><td></td></tr><tr><td> MgCl 2 </td><td> 시그마 - 알드리치 </td><td> M - 8266 </td><td></td></tr><tr><td> 자당 </td><td> 시그마 - 알드리치 </td><td> 16,104 </td><td></td></tr><tr><td> 트립신 / EDTA (에틸렌 다이아 민 테트라 초산) 솔루션 </td><td> 시그마 - 알드리치 </td><td> T4174 </td><td></td></tr><tr><td> 피펫 팁 </td><td> 사용자가 만든 </td><td></td><td></td></tr><tr><td> 전기 펄스 발생기 </td><td> 사용자가 만든 </td><td></td><td></td></tr><tr><td> 6 웰 플레이트 </td><td> TPP </td><td> 92,406 </td><td></td></tr><tr><td> 15 ML의 원심 분리기 튜브 </td><td> TPP </td><td> 91,015 </td><td></td></tr><tr><td> 75cm 두 문화 술병 </td><td> TPP </td><td> 90,076 </td><td></td></tr></tbody></table>

changing the direction and orientation of electric field during electric pulses application improves plasmid gene transfer in vitro

진 electrotransfer 작은 분자로 그 침투성하고, 세포막의 불안정의 원인이 짧고 강렬한 전기 펄스의 응용 프로그램에 의해 세포에 유전자를 전달하기 위해 사용하는 물리적 방법이고 이러한 DNA와 같은 큰 분자의 전송을 허용합니다. 이것은 안전성, 효능 및 응용 프로그램의 용이성으로 인해 바이러스 벡터에 대한 대안을 나타냅니다. 유전자 electrotransfer에 대해 다른 전기 펄스 프로토콜은 그 중 하나가 펄스 전송 중에 전기장 방향과 방향을 변화, 최대 유전자 transfection을 달성하기 위해 사용됩니다. 전기장 방향과 방향을 변경하면 세포에 DNA 항목에 대한 관할 멤브레인 영역을 향상시킬 수 있습니다. 이 비디오에서는, 우리는 모든 펄스가 같은 방향으로 전달되며 펄스가 또는 전기장의 방향과 방향을 변경하여 전달하는 경우 유전자 electrotransfer의 효율에 차이를 보여줍니다. 통합 전극과 전기 펄스 응용 프로그램을하는 동안 다른 방향으로 전기장의 변화를 수있는 고전압 프로토 타입 생성기,이 목적 팁 경우, 사용되었습니다. 진 electrotransfer의 효험은 모든 세포의 숫자로 나눈 녹색 형광 단백질을 표현 세포의 숫자와 같은 펄스 신청 후 24 시간 결정됩니다. 결과는 전기 펄스 전송 중에 전기장의 방향이 바뀌면 유전자 transfection이 증가는 것을 보여줍니다.

Watch this Scientific Journal Video about Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro at JoVE.com

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

전기장의 방향이 펄스 응용 프로그램 중에 변경되면 electroporation에 의해 유전자 transfection이 약 두 배 향상이며, 동시에 세포 생존 능력은 영향을받지 않습니다. 유전자 transfection의 증가는 세포로 DNA의 항목에 대해 관할 만들어 막 면적의 증가로 인해 발생할 수 있습니다.

전기 펄스 응용 프로그램에는 방향과 전기장의 방향을 변경하면 플라스미드 유전자 전송을 향상 체외에서</em

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause ...

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Medicine

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

10.4K 조회수.  University of Ljubljana. 전기장의 방향이 펄스 응용 프로그램 중에 변경되면 electroporation에 의해 유전자 transfection이 약 두 배 향상이며, 동시에 세포 생존 능력은 영향을받지 않습니다. 유전자 transfection의 증가는 세포로 DNA의 항목에 대해 관할 만들어 막 면적의 증가로 인해 발생할 수 있습니다.

비디오: Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

면역 결함 마우스의 확립과 인간 Retinoblastoma 종양의 전파

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

연구

의학

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

잠복기 모델에서 허혈성 심장 마비에 대한 유전자 전송

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

마우스에 영구 중간 대뇌 동맥 내고의 응용

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

암의 생산 및 반응 산소 종의 감지 (ROS)

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

PTSD의, 신경 Hematologic, 그리고 행동 상관 관계를 누설하는 행위에 대해서는 동물 모델에서 생체

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

가수 분해 크로스 링커를 통해 아데노 바이러스 벡터 닿는를 사용하여 금속 스텐​​트 표면에서 혈관 유전자 전송

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

소설<em&gt; 생체</em&gt; 유전자 전달 기술과<em&gt; 시험관</em근골격계 질환의 뼈 손실의 연구를위한&gt; 셀 기반 분석 실험

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

인간 종양 이종 이식에서 마우스 세포의 고갈은 크게 대상 세포의 다운 스트림 분석을 향상

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

현미경 기반 방법을 생체 외에서 상처 치유 중 상피 세포 이동의 평가 대 한

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

Vivo에서 토끼 일반적인 경 동맥 내 피에 유전자 이동

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...