이 작품은 NIH에서 교부금에 의해 지원되었습니다 R03 DK089061-01 (VKY), NIH : K08 DK068391 (VKY), 당뇨병과 내분비학 연구 센터 - (DERC - P30DK079638) 의학 베일러 대학에서 파일럿 및 타당성 부여 DERC (VKY), 아동 당뇨병 연구 재단 : JDRF 상 # 5-2006-134 (VKY).

<ol>
	<li>Parks, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+helper-dependent+adenovirus+vector+system:+removal+of+helper+virus+by+Cre-mediated+excision+of+the+viral+packaging+signal.">A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal.</a> Proc. Natl. Acad. Sci. U. S. A. 93, 13565-13570 (1996).</li><li>Kim, I. H., Jozkowicz, A., Piedra, P. A., Oka, K., Chan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifetime+correction+of+genetic+deficiency+in+mice+with+a+single+injection+of+helper-dependent+adenoviral+vector.">Lifetime correction of genetic deficiency in mice with a single injection of helper-dependent adenoviral vector.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 13282-13287 (2001).</li><li>Belalcazar, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stable+expression+of+human+apolipoprotein+A-I+mediated+by+helper-dependent+adenovirus+gene+transfer+inhibits+atherosclerosis+progression+and+remodels+atherosclerotic+plaques+in+a+mouse+model+of+familial+hypercholesterolemia.">Long-term stable expression of human apolipoprotein A-I mediated by helper-dependent adenovirus gene transfer inhibits atherosclerosis progression and remodels atherosclerotic plaques in a mouse model of familial hypercholesterolemia.</a> Circulation. 107, 2726-2732 (2003).</li><li>Oka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stable+correction+of+low-density+lipoprotein+receptor-deficient+mice+with+a+helper-dependent+adenoviral+vector+expressing+the+very+low-density+lipoprotein+receptor.">Long-term stable correction of low-density lipoprotein receptor-deficient mice with a helper-dependent adenoviral vector expressing the very low-density lipoprotein receptor.</a> Circulation. 103, 1274-1281 (2001).</li><li>Nomura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-density+lipoprotein+receptor+gene+therapy+using+helper-dependent+adenovirus+produces+long-term+protection+against+atherosclerosis+in+a+mouse+model+of+familial+hypercholesterolemia.">Low-density lipoprotein receptor gene therapy using helper-dependent adenovirus produces long-term protection against atherosclerosis in a mouse model of familial hypercholesterolemia.</a> Gene Ther. 11, 1540-1548 (2004).</li><li>Ng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-efficiency+Cre/loxP-based+system+for+construction+of+adenoviral+vectors.">A high-efficiency Cre/loxP-based system for construction of adenoviral vectors.</a> Hum. Gene Ther. 10, 2667-2672 (1999).</li><li>Palmer, D., Ng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+system+for+helper-dependent+adenoviral+vector+production.">Improved system for helper-dependent adenoviral vector production.</a> Mol. Ther. 8, 846-852 (2003).</li><li>Oka, K., Chan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helper-Dependent+Adenoviral+Vectors.">Helper-Dependent Adenoviral Vectors.</a> Current Protocols in Molecular Biology. , 16.24.1-16.24.23 (2005).</li><li>Yechoor, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenin3+is+sufficient+for+transdetermination+of+hepatic+progenitor+cells+into+neo-islets+in+vivo+but+not+transdifferentiation+of+hepatocytes.">Neurogenin3 is sufficient for transdetermination of hepatic progenitor cells into neo-islets in vivo but not transdifferentiation of hepatocytes.</a> Dev. Cell. 16, 358-373 (2009).</li><li>Yechoor, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Therapy+with+Neurogenin+3+and+Betacellulin+Reverses+Major+Metabolic+Problems+in+Insulin-Deficient+Diabetic+Mice.">Gene Therapy with Neurogenin 3 and Betacellulin Reverses Major Metabolic Problems in Insulin-Deficient Diabetic Mice.</a> Endocrinology. , (2009).</li></ol>

관심 없음 충돌이 선언 없습니다.

HDAds는 초기 세대 광고의 약점을 극복하고 유전자 치료 응용 프로그램에 대한 활용하기 위해 개발되었습니다. 그러나, 기술적 인 문제가 남아 있습니다. 예를 들어, HDAd는 HDAd의 포장 및 벡터 증폭을위한 HV는 초기 세대 광고만큼 효율적이지 않습니다 필요합니다. HV는 1 세대 광고 및 HV 타협 HDAd의 효과의 오염입니다. 따라서, 고효율 transfection 각 직렬 통과에 대한 최적의 조건 중요합니다. 벡터 생산을위한 또 다른 중요한 매개 변수는 직접 정지 전지 inoculum로 사용됩니다 이후의 통로 5 사용되어야하는 통로 (P1-P4)입니다. 우리의 경험으로, 가장 좋은 결과는 HDAd 벡터 비율은 크게 다음과 같은 통로 (그림 2의 P3)에 증가되는이 통로를 사용하여 얻을 수 있습니다. HDAd 벡터의 수율은 transgene 카세트에 따라 달라집니다. 두 유전자가 아래에 있기 때문에 벡터 생산하는 동안 두 transgenes이 표현됩니다유비쿼터스 발기인. Ngn3는 전사 인자이며, Btc는 표현 성장 호르몬은 벡터 복제 및 포장에 도움이 동안 세포 계보 (Lineage)는 벡터 증폭을 억제 영향을 미칠 수 있다는 HDAd 벡터 표현 전사 인자를 제안 성장 인자입니다. 당뇨병은 전염병 비율을 가정으로, B-세포 질량을 복원 할 수있는 새로운 접근 방법이 필요합니다. 이 보고서에서, 우리는 작은 섬 성장 인자와 함께 작은 섬 계보 (Lineage) - 정의 전사 인자, Ngn3의 유전자 전달에 영향을 미치도록 HDAd 벡터의 장점을 활용하는 방법을 설명, betacellulin은 간 periportal 지역에서 작은 섬의 neogenesis를 유도합니다. 이 효능을 평가하기 위해, 그것은 안정 고혈당증있는 마우스를 선택하고 적절한 컨트롤이 항상 포함되어 있는지 확인하는 것이 중요합니다. 이 유전자 전송 실험 빈 벡터는 당뇨병 마우스는 항상 활용해야 처리. 또한, HDAd - Ngn3 및 HDAd-Btc를 사용하여 개별적으로 당뇨병 MI에게 치료를CE는 작은 섬 neogenesis에서이 두 유전자의 개별 기여를 테스트하는 역할을한다. 우리의 데이터는 혼자 Ngn3이 작은 섬 neogenesis를 유도하기에 충분하지만, 성장 인자, Btc의 추가는 작은 섬 neogenesis의 강력한 유도로 이어지는 응답을 증가하는 역할을 증명 있습니다. 이 벡터 표현은 실제로 대상 조직, 간에서 달성하고 있으며, 치료를 생쥐의 플라즈마에서 assayed 인슐린은 췌장의 부재를 보여주는하여 췌장의 잔류 섬에서 오는되지 않습니다 있다는 것을 입증하는 것을 테스트하는 것도 중요합니다 당뇨병 쥐 섬. 요약하면, 유전자 전송에 대한 HDAd - 벡터 시스템의 장점은 호스트에 최소 만성 독성뿐만 아니라 벡터 게놈의 비 통합의 자연과 간에서 높은 복제 능력, 효율적인 전달과 긴 지속적인 유전자 발현에 있습니다 염색체. 기본 제한은 세대와 관련된 복잡한 단계의생체 응용 프로그램에서 주로 가장 인기있는 광고 혈청 형 5 간으로 제한됩니다. 작은 섬 neogenesis 완전히 작은 섬 성장 인자, betacellulin과 함께 작은 섬 계보 (Lineage) - 정의 전사 인자, Ngn3의 유전자 전달에 의해 간에서 췌장의 neogenesis을 유도하여 플라즈마 인슐린과 당뇨병 쥐에서 포도당 내성을 복원하도록 유도 할 수 있습니다. 이 보고서에서, 우리는 고품질 HDAd - Ngn3 및 HDAd-Btc를 생성하는 최적의 프로토콜을 표시하고, 고혈당증을 반대로 당뇨병 생쥐의 간에서 작은 섬 neogenesis를 유도하고 평가하는 기술을 보여줍니다. 각주 : 바이러스 성 벡터와 세포 라인이 여기서 설명은 벡터 생산 핵심 연구소, 당뇨병 연구 센터, 의학 베일러 대학 (에서 구입하실 수 있습니다 <a href="http://www.bcm.edu/mcb/index.cfm?pmid=7731" target="_blank">http://www.bcm.edu/mcb/index.cfm?pmid=7731</a> ). 일부 상용 키트 HDAd 바이러스 (예를 들면 Microbix biosyst 생성하기 위해 또한 이용 가능합니다EMS 주식회사).

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> ProFectionR 포유류의 Transfection 키트 </td><td> Promega </td><td> E1200 </td></tr><tr><td> DNeasy 혈액 및 조직 키트 (50) </td><td> Qiagen </td><td> 69,504 </td></tr><tr><td> PerfeCTa SYBR 그린 SuperMix, ROX </td><td> 콴타 Biosciences </td><td> 95055-500 </td></tr><tr><td> 나트륨 deoxycholate </td><td> 시그마 </td><td> D6750-25g까지 </td></tr><tr><td> 가상 분말 </td><td> Invitrogen </td><td> 61100087 </td></tr><tr><td> 페니실린의 스트렙토 마이신 </td><td> 시그마 </td><td> 15140122 </td></tr><tr><td> FBS </td><td> 애틀랜타 체액 </td><td> S11150 </td></tr><tr><td> L-글루타민 </td><td> Invitrogen </td><td> 25030-081 </td></tr><tr><td> Hygromycin B </td><td> 시그마 </td><td> H0654-1G </td></tr><tR&gt; <td> 가상 EAGLE JOKLIK </td><td> 시그마 </td><td> M0518-10L </td></tr><tr><td> Rnase </td><td> 로슈 </td><td> 10109169001 </td></tr><tr><td> DNase I, 등급 II </td><td> 로슈 </td><td> 10104159001 </td></tr><tr><td> Streptozocin </td><td> 시그마 </td><td> s0130 </td></tr><tr><td> 유리 스피너 플라스크 </td><td> 코닝 </td><td> 4500-3L </td></tr><tr><td> 유리 스피너 플라스크 </td><td> 코닝 </td><td> 4500-250 </td></tr><tr><td> A-lyzer casset 슬라이드 </td><td> 붙여 CH </td><td> PI66380 </td></tr><tr><td> 튜브 optiseal 폴리 allomer, 11.2 ML </td><td> Beckman 보습 바로 앞에 달린 풀 베는 날 </td><td> 362181 </td></tr><tr><td> 세슘 염화 1kg </td><td> JT4042-2 </td><td> VWR </td></tr><tr><td> Beckman LE-80K </td><td> Beckman 보습 바로 앞에 달린 풀 베는 날 </td><td> 최적의 LE-80K ultracentrifuge </td></tr><tr><td> 필터 </td><td> VWR </td><td> 28143-338 </td></tr><tr><td> 관 500m를 원심 분리기리터 </td><td> 코닝 </td><td> 431123 </td></tr><tr><td> tailveiner restrainer </td><td> Braintree 과학, INC </td><td> TV-150 </td></tr><tr><td> 인슐린, 마우스 엘리사 </td><td> Mercodia </td><td> 10-1247-01 </td></tr><tr><td> microvette CB300 </td><td> Sarstedt </td><td> 16.443.100 </td></tr><tr><td> D-글루코오스 </td><td> 시그마 </td><td> G8270 </td></tr><tr><td> 마우스 C-펩타이드 엘리사 키트 </td><td> 와코 순수 화학 산업 (주) </td><td> # 631-07231 </td></tr><tr><td> 기니 돼지 방지 인슐린 항체 </td><td> Abcam </td><td> ab7842 </td></tr><tr><td> 염소 안티 - Pdx1 항체 </td><td> 박사 크리스토퍼 라이트 선물 </td><td></td></tr><tr><td> 마우스 방지 Ngn3 항체 </td><td> 베타 세포 생물학 컨소시엄, 대학. 펜실베니아 </td><td> AB2013 </td></tr><tr><td> 마우스 방지 Nkx6.1 항체 </td><td> 베타 세포 생물학 컨소시엄, 대학. Pennsy의lvania </td><td> F64A6B4 </td></tr><tr><td> 안티 betacellulin 항체 </td><td> 세포 과학 </td><td> PAAQ1 </td></tr><tr><td> ALT (SGPT) 컬러 시약 SET. </td><td> Teco 진단 </td><td> A526 - 120 </td></tr><tr><td> 대서양 표준시 / (SGOT), 색상 엔드 포인트 시약 세트 </td><td> Teco 진단 </td><td> A561-120 </td></tr></tbody></table> 표 2. 특정 시약 및 장비.

neo-islet formation in liver of diabetic mice by helper-dependent adenoviral vector-mediated gene transfer

1 형 당뇨병은 췌장의 인슐린 생산 세포의 T 세포 매개 면역 파괴에 의해 발생합니다. 작은 섬 이식은 기증자의 가용성에 의해 및 장기 immunosuppression의 필요성에 의해 제한 되었기 때문에 지금까지 인슐린 대체 여전히 주요 치료입니다. Neuogenin3의 유전자 전송 (Ngn3), 작은 섬 계보 (Lineage) - 정의 특정 전사 인자와 Betacellulin (Btc)에 의해 유도 작은 섬 neogenesis는 작은 섬 성장 인자는 제 1 형 당뇨병을 치료 할 수있는 가능성이 있습니다. Adenoviral 벡터 (광고) 고효율 유전자 전달 벡터이다;하지만, 초기 세대 광고 생체 사용하는 몇 가지 단점이 있습니다. 도우미에 의존 광고 (HDAds) 광고의 조기 세대의 안전 프로필을 향상시키고 transgene 표현 한을 연장하기 위해 개발 된 가장 진보 된 광고입니다. 그들은 바이러스 코딩 시퀀스에게 2-5 부족하며 광고 CIS 엘을 유지하기 때문에 만성 독성이 부족벡터 복제 및 포장에 필요한 ements. 이 36 이하 유전자까지의 복제를 할 수 있습니다. 이 프로토콜에서는, 우리는 HDAd - Ngn3 및 HDAd-Btc를 생성하고 STZ 유발 당뇨병 마우스에이 벡터를 제공하는 방법을 설명합니다. 우리의 결과는 HDAd-Ngn3, 간에서 HDAd - Btc 유도 &#39;네오 섬&#39;의 공동 주입하고하면 당뇨병 생쥐에서 고혈당증을 반대로 것을 보여줍니다.

Watch this Scientific Journal Video about Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer at JoVE.com

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

우리는 간장 네오 작은 섬 STZ의 형성 (streptozotocin) 유도 도우미에 의존 adenoviral 벡터 (HDAd)과 고혈당증의 반전을 사용하여 유전자 Neurogenin3의 전송 (Ngn3)와 Betacellulin (Btc)에 의해 당뇨병 쥐를.을 설명 우리의 방법은 생체 전달하고 오래 지속 유전자 발현에서의 고효율과 도우미에 의존 adenoviral 벡터의 장점이 소요됩니다.

도우미에 의존 Adenoviral 벡터로 인한 유전자의 전송에 의한 당뇨 쥐의 간에서 네오 - 작은 섬 형성

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

neo-islet-formation-liver-diabetic-mice-helper-dependent-adenoviral

Research

JoVE Journal

Medicine

11.7K 조회수.  Baylor College of Medicine. 우리는 간장 네오 작은 섬 STZ의 형성 (streptozotocin) 유도 도우미에 의존 adenoviral 벡터 (HDAd)과 고혈당증의 반전을 사용하여 유전자 Neurogenin3의 전송 (Ngn3)와 Betacellulin (Btc)에 의해 당뇨병 쥐를.을 설명 우리의 방법은 생체 전달하고 오래 지속 유전자 발현에서의 고효율과 도우미에 의존 adenoviral 벡터의 장점이 소요됩니다.

비디오: Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Use of a Hanging-weight System for Liver Ischemia in Mice

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate1-2. Therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies3-9. Specifically, conditional mice with tissue specific deletion of a gene (cre, flox system) provide insights into the role of proteins in particular tissues10-13 . Because of the technical difficulty associated with manually clamping the portal triad in mice, we performed a systematic evaluation using a hanging-weight system for portal triad occlusion which has been previously described3. By using a hanging-weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes, since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels. Furthermore, the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved. Furthermore, the portal triad is only manipulated once throughout the entire surgical procedure. As a result, procedures like pre-conditioning, with short times of ischemia and reperfusion, can be easily performed. Systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels3,9. Taken together, these studies confirm highly reproducible liver injury when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Thus, this technique might be useful for other investigators interested in liver ischemia studies in mice. Therefore the video clip provides a detailed step-by-step description of this technique.

We established a novel murine model of a hanging weight system for portal triad occlusion. This technique may be useful for future investigations of ischemia in murine hepatic models.

마우스의 간 국소 빈혈에 대한 교수형 - 체중 시스템의 사용

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and ...

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의학

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 ...

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 ...

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used techniques require killing of the animals and postmortem recovery of the meal1,2. This approach prevents longitudinal studies to determine changes in gastric emptying with age and progression of disease. The commonly used [13C]-octanoic acid breath test for humans3 has been modified for use in mice4-6 and rats7 and we previously showed that this test is reliable and responsive to changes in gastric emptying in response to drugs and during diabetic disease progression8. In this video presentation the principle and practical implementation of this modified test is explained. As in the previous study, NOD LtJ mice are used, a model of type 1 diabetes9. A proportion of these mice develop the symptoms of gastroparesis, a complication of diabetes characterized by delayed gastric emptying without mechanical obstruction of the stomach10.
This paper demonstrates how to train the mice for testing, how to prepare the test meal and obtain 4 hr gastric emptying data and how to analyze the obtained data. The carbon isotope analyzer used in the present study is suitable for the automatic sampling of the air samples from up to 12 mice at the same time. This technique allows the longitudinal follow-up of gastric emptying from larger groups of mice with diabetes or other long-standing diseases.

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice.

를 사용하여 비 비만 당뇨병 마우스의 위 비우는의 평가 [<sup&gt; 13</sup&gt; C]-octanoic 산성 호흡 시험

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used ...

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 ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

마우스의 담관 결찰 : 폐쇄성 담즙에 의한 염증성 간 손상과 섬유화의 유도

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 &#176;C.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 &#176;C).

Subnormothermic 기계 관류의 수단으로 기능 인간의 간 보존 및 복구

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

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 ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

당뇨병 마우스에서 만성 상처를 만드는 프로토콜

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

돼지에서 자가 이식 Hepatocytes 전 비보 의 lentiviral 벡터 중재 된 유전자 치료

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...