이 작품은 국립 보건원 (7R01HL098528-03)와 FAMRI의 임상 혁신 상 수상에 의해 지원되었다.

1. 단백질 형질 시약의 준비 <ol><li> 드라이 필름을 포함하는 튜브에 메탄올 또는 클로로포름 250 μL를 추가하여 프로 피사체 시약을 녹인다. </li><li> 최고 속도로 20 초 동안 소용돌이. </li><li> 별도의 microcentrifuge 튜브에 프로 피사체 시약의 피펫 20 μL. </li><li> 실온에서 6 시간의 최소 층류 후드 아래 프로 피사체 시약을 포함하는의 microcentrifuge 튜브를 배치하여 용매를 증발. 그것은 완전히 건조해야합니다. 또한, 진공 건조기를 사용하여 프로 피사체 시약을 건조. </li><li> -20 ° C.에 튜브를 저장 그들은이 온도에서 일년 좋다. </li></ol> 2. 프로 피사체 시약에 단백질을 추가 <ol><li> 건조 프로 피사체의 형질 시약 20 μL를 포함하는 관으로​​, 관심의 단백질 2.0 μg을 함유 PBS의 피펫 50 μL. </li><li> 단백질을 추가 한 후, 저속 F에서 소용돌이로 교반하여 용액을 혼합또는 3-5 초 실온에서 30 분 동안 배양 한 다음. </li></ol> 3. 단백질의 기관지 관리 <ol><li> 케타민을 / xylazine의 복강 내 주사 (100 ㎎ / ㎏, 10 ㎎ / ㎏)로 마우스를 마취. </li><li> 진정 작용이 달성 된 후, 마우스는 특별히 디자인 된 플랫폼 (그림 1)를 사용하여 상단 incisors에서 일시 중단됩니다. </li><li> 집게와 후두경으로 종이 클립 사용, 구강 인두는 카테터 (그림 2) 도입 할 수 있도록 열립니다. 성대는 마우스의 기관에 광원을 배치하여 시각입니다. </li><li> 단백질 / 프로젝트 시약 혼합물 (50 μL)는 펜 세기 Microsprayer을 사용하여 기관을 통해 주입된다. </li></ol>

<ol>
	<li>Glasser, S. W., Korfhagen, T. R., Wert, S. E., Whitsett, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+models+for+study+of+pulmonary+development+and+disease.">Transgenic models for study of pulmonary development and disease.</a> Am. J. Physiol. 267, 489-497 (1994).</li><li>D'Armiento, J., Dalal, S. S., Okada, Y., Berg, R. A., Chada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagenase+expression+in+the+lungs+of+transgenic+mice+causes+pulmonary+emphysema.">Collagenase expression in the lungs of transgenic mice causes pulmonary emphysema.</a> Cell. 71, 955-961 (1992).</li><li>Foronjy, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+dismutase+expression+attenuates+cigarette+smoke-+or+elastase-generated+emphysema+in+mice.">Superoxide dismutase expression attenuates cigarette smoke- or elastase-generated emphysema in mice.</a> Am. J. Respir Crit. Care Med. 173, 623-631 (2006).</li><li>Foronjy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+divergent+roles+of+secreted+frizzled+related+protein-1+(SFRP1)+in+lung+morphogenesis+and+emphysema.">The divergent roles of secreted frizzled related protein-1 (SFRP1) in lung morphogenesis and emphysema.</a> Am. J. Pathol. 177, 598-607 (2010).</li><li>Costa, R. H., Kalinichenko, V. V., Lim, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors+in+mouse+lung+development+and+function.">Transcription factors in mouse lung development and function.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 280, 823-838 (2001).</li><li>Perl, A. K., Tichelaar, J. W., Whitsett, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+expression+in+the+respiratory+epithelium+of+the+mouse.">Conditional gene expression in the respiratory epithelium of the mouse.</a> Transgenic Research. 11, 21-29 (2002).</li><li>Morimoto, M., Kopan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=rtTA+toxicity+limits+the+usefulness+of+the+SP-C-rtTA+transgenic+mouse.">rtTA toxicity limits the usefulness of the SP-C-rtTA transgenic mouse.</a> Dev. Biol. 325, 171-178 (2009).</li><li>Waehler, R., Russell, S. J., Curiel, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+targeted+viral+vectors+for+gene+therapy.">Engineering targeted viral vectors for gene therapy.</a> Nature reviews. Genetics. 8, 573-587 (2007).</li><li>Crystal, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+an+adenovirus+containing+the+human+CFTR+cDNA+to+the+respiratory+tract+of+individuals+with+cystic+fibrosis.">Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis.</a> Nat. Genet. 8, 42-51 (1994).</li><li>Merkel, O. M., Zheng, M., Debus, H., Kissel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+gene+delivery+using+polymeric+nonviral+vectors.">Pulmonary gene delivery using polymeric nonviral vectors.</a> Bioconjugate Chemistry. 23, 3-20 (2012).</li><li>Yang, Y., Li, Q., Ertl, H. C., Wilson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+humoral+immune+responses+to+viral+antigens+create+barriers+to+lung-directed+gene+therapy+with+recombinant+adenoviruses.">Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses.</a> J. Virol. 69, 2004-2015 (1995).</li><li>Liu, Q., Muruve, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+the+inflammatory+response+to+adenovirus+vectors.">Molecular basis of the inflammatory response to adenovirus vectors.</a> Gene Ther. 10, 935-940 (2003).</li><li>Pfeifer, C., Aneja, M. K., Hasenpusch, G., Rudolph, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+serotype+9-mediated+pulmonary+transgene+expression:+effect+of+mouse+strain,+animal+gender+and+lung+inflammation.">Adeno-associated virus serotype 9-mediated pulmonary transgene expression: effect of mouse strain, animal gender and lung inflammation.</a> Gene Ther. 18, 1034-1042 (2011).</li><li>Wallace, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+phosphatase+2A+regulates+innate+immune+and+proteolytic+responses+to+cigarette+smoke+exposure+in+the+lung.">Protein phosphatase 2A regulates innate immune and proteolytic responses to cigarette smoke exposure in the lung.</a> Toxicol. Sci. 126, 589-599 (2012).</li><li>Wallace, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Phosphatase+2a+(Pp2a)+Regulates+Innate+Immune+and+Proteolytic+Responses+to+Cigarette+Smoke+Exposure+in+the+Lung.">Protein Phosphatase 2a (Pp2a) Regulates Innate Immune and Proteolytic Responses to Cigarette Smoke Exposure in the Lung.</a> Toxicol. Sci. , (2012).</li><li>Brown, R. H., Walters, D. M., Greenberg, R. S., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+endotracheal+intubation+and+pulmonary+functional+assessment+for+repeated+studies+in+mice.">A method of endotracheal intubation and pulmonary functional assessment for repeated studies in mice.</a> J. Appl. Physiol. 87, 2362-2365 (1999).</li></ol>

저자는 공개 할 관심 없음 충돌이 없습니다.

다른 방법에 비해이 기술의 장점은 폐 조직 자체 내에서 단백질 수준과 활동의 증가를 생성하는 것입니다. 또한, 그것은 단지기도 내에 체류 반대로 폐의 가장 말단 지역에 침투합니다. 우리는 심지어 생리 식염수 15기도 lavaging 후 injectate 증가 단백질의 활동을 측정했다. 조직 활동 분석은 단백질이 세포를 입력하고 단지 마우스의 공​​기 공간에 남아 있지 않는 것을 나타냅니다. 이 또?...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Pro-Ject</td>
 <td>Pierce Bio</td>
 <td>89850</td>
 <td></td>
 </tr>
 <tr>
 <td>Microsprayer</td>
 <td>Penn Century</td>
 <td>FMJ-250</td>
 <td></td>
 </tr>
 <tr>
 <td>Beta-galactosidase</td>
 <td>Pierce Bio</td>
 <td>89850</td>
 <td></td>
 </tr>
 <tr>
 <td>Beta-galactosidase antibody</td>
 <td>Santa Cruz Bio</td>
 <td>SC-19119</td>
 <td></td>
 </tr>
 <tr>
 <td>Mouse serum albumin</td>
 <td>Sigma Aldrich</td>
 <td>A3139</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine/xylazine</td>
 <td>Sigma Aldrich</td>
 <td>K113</td>
 <td></td>
 </tr>
 </tbody>
</table>

protein transfection of mouse lung

단백질 발현을 증가하는 것은 연구자가 더 중요한 생물 학적 과정 1 조절에 그 단백질의 기능 역할을 이해 할 수 있습니다. 폐에서이 유전자 발현을 증가 4를 통해 단백질 수준을 상승 형질 전환 생쥐 2,3 또는 바이러스 성 또는 비 바이러스 성 벡터를 활용 유전 방식을 통해 일반적으로 달성되었다. 형질 전환 생쥐를 생성하는 비용과 시간이 많이 소요하고 유전자 또는 만성 유전자 발현의 임의 삽입은 정상 폐 발달을 변경하고 따라서 모델 5의 유틸리티를 제한 할 수 있습니다. 조건식을 생성하는 데 사용되는 만성 유전자 발현 6, 반대 테트라 사이클린 제어 transactivator (rtTA) 마우스와 관련된 문제 AVERT 조건부 형질 전환은 자연 공기 공간 확대 7을 개발하면서. 형질 전환과 마찬가지로, 바이러스 성 및 비 바이러스 성 벡터의 사용은 8 비싸고 용량-D를 자극 할 수그 ependent 염증 반응 결과 9 혼란과 표현 10을 방해. 또한, 반복 투여의 효능은 벡터 11,12 향상 면역 반응에 의해 제한됩니다. 연구진은 적은 염증을 유발하고 폐 13에서 더 이상 표현이 아데노 - 관련 바이러스 (AAV) 벡터를 개발하고 있습니다. β-갈 락토시다 아제를 사용하여, 우리는 직접 단백질 형질 전환 기술을 사용하여 폐 내에서 신속하고 효율적으로 증가하는 단백질 발현을위한 방법을 제시한다. 이 프로토콜은 폐 조직 자체에 침투를 허용하는 지질 기반 형질 시약 20 μL (프로 피사체, 피어스 바이오)와 단백질을 정제 고정 된 금액을 섞는다. 리포좀 단백질 혼합물은 microsprayer (펜 세기, 필라델피아, PA)를 사용하여 기관을 통해 생쥐의 폐에 주입된다. microsprayer은 폐를 통해 액체 에어로졸의 벌금 깃털을 생성합니다. 기술을 사용하여우리는기도와 마우스 (14)의 폐포에 걸쳐 주입 단백질의 균일 한 증착을 증명하고있다. 지질 형질 전환 기술은 효과를 달성하기 위해 단백질의 소량 사용할 수 있습니다. 이 그렇지 않은 고단백 관리에 의해 자극 될 것이다 염증 반응을 제한합니다. 사실,이 기술 사용하여 우리는 우리가 크게 폐 세척 세포질 15에 영향을주지 않고 폐에 PP2A 활동을 증가시킬 수 있다고 발표했다. 공격 후 24 시간을 촬영 한 폐 세척의 세포질은 컨트롤에 비교했다 (27 ± 31 대 4 제어 ± 5 알부민, 형질 전환, N 그룹 당 = 6). 또한, 형질 전환 모델에서 발생할 수있는 폐 발달 변경이나 아키텍처 변경을 유도하지 않고 단백질 수준을 증가시킵니다. 그러나 반복 투여의 필요성 단백질 발현에 장기적인 증가의 효과를 조사 연구를위한이 기술은 불리한 만들 수 있습니다. 특히 트루 것짧은 반감기를 가진 단백질에 대한 전자.

<html> 우리의 기술의 효과를 입증하기 위해 우리는 프로 피사체의 형질 시약 20 μL를 포함 PBS의 50 μL에 녹인 마우스 2 μg 알부민 (시그마)와 생쥐의기도를 주입하는 microsprayer (펜 세기)를 사용 하였다. 알부민 치료 마우스는 베타 - 갈 락토시다 아제 단백질의 2 μg (피어스 바이오)와 동일한 방법으로 처리 하였다 생쥐와 비교 하​​였다. 이십사시간 후, 마우스는 안락사되었고 폐 조직 학적 분석을 ?...

Watch this Scientific Journal Video about Protein Transfection of Mouse Lung at JoVE.com

Protein Transfection of Mouse Lung

형질 전환 마우스 또는 바이러스 성 벡터는 폐 내의 단백질 발현을 증가하는 데 사용되었습니다. 그러나이 기술은 기술적으로 도전적인 시간이 소요하고 결과를 혼동 할 수 있습니다 떨어져 표적 장기에 주요 영향이있다. 우리의 단백질 형질 프로토콜은 지질 기반의 transfection 시약 및 균일 폐 세포에 활성 단백질을 제공하는 초 미세 microsprayer을 사용합니다.

마우스 폐의 단백질 형질

Increasing protein expression enables researchers to  better understand the functional role of that protein in regulating key  biological processes1. In ...

Research

JoVE Journal

Biology

9.3K 조회수.  St. Luke's Roosevelt Medical Center. 형질 전환 마우스 또는 바이러스 성 벡터는 폐 내의 단백질 발현을 증가하는 데 사용되었습니다. 그러나이 기술은 기술적으로 도전적인 시간이 소요하고 결과를 혼동 할 수 있습니다 떨어져 표적 장기에 주요 영향이있다. 우리의 단백질 형질 프로토콜은 지질 기반의 transfection 시약 및 균일 폐 세포에 활성 단백질을 제공하는 초 미세 microsprayer을 사용합니다.

비디오: Protein Transfection of Mouse Lung

Gene-gun Transfection of Hippocampal Neurons

Hi, I'm Kma. I work in Case Lab at Brown University Clean Slicer stage with 70%ethanol. Place plate insert into six.Well plate with medium and incubate at 37 degree whole brain with tweezers between midbrain and four brainin. Isolate hippocampus on one side, isolate hippocampus from the second side. Place hippocampi onto A PBS drop.Aspirate out the PBS make sagittal sections using the tissue slicer. Repeat transfer slicers onto a perish by squatting with PBS wash sections by adding and aspirating PBS and medium gently. Separate sections with tweezers.Wash sections with PBS and media. Again, transfer sections onto the plate and insert. Aspirate out the media in sections using tweezers.Attach gene gun hose to the helium cylinder. Attach barrel to gene gun. Fill the magazine with bullets.Insert magazine into the Jing gun. Adjust the pressure on the helium tank shoot. EGFP coated beads onto the sections.And now.

Hippocampal 뉴런의 유전자 총을 Transfection

연구

생물학

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

액상 복구와 분자량 기반 분류에 대한 GELFREE 8100 분획화 시스템을 사용하여

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Direct Tracheal Instillation of Solutes into Mouse Lung

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice. Using this approach, one can target delivery of test solutes or solids (such as lung therapeutics, surfactants, viruses, and small oligonucleotides) into the distal lung. Tracheal instillations may be the preferred methodology, over inhalation protocols that may primarily target the upper respiratory tract and possibly expose the investigator to potentially hazardous substances. Additionally, in using the tracheal instillation protocol, animals can fully recover from the non-invasive procedure. This allows for making subsequent physiological measurements on test animals, or reinstallation using the same animal. The amount of instillate introduced into the lung must be carefully determined and osmotically balanced to ensure animal recovery. Typically, 30-75 &mu;L instillate volume can be introduced into mouse lung.

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice.

마우스 폐에 Solutes 직접 Tracheal 떨어뜨림

Intratracheal instillations deliver solutes directly into the lungs.  This procedure targets the delivery of the instillate into the distal regions of the ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

마우스 배아의 폐 문화, 분기의 분자 메커니즘을 평가하는 시스템

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

생체내 이미징 및 난소암의 Orthotopic 마우스 모델에서 치료 트리 트먼트

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

칙 태아의 단일 세포 Transfection

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

절연, 환기 및기구 마우스 폐의 특성화는 타악기 흐름 Perfused

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Hoechst의 분리 및 특성 낮은 CD45 부정적인 마우스 폐 Mesenchymal 줄기 세포

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

유도 만능의 줄기 세포에서 파생 마우스의 생성

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...