이 작품은 국립 과학 재단 (National Science Foundation)의 경력 수상 (CBET - 0846395)과 비교 의학의학과 툴란 대학에 의해 투자되었다.

1. 동물 <ol><li> 여성 C57BL/6J 생쥐 (뮤스 musculus)는 전에 출하하는 공급 업체에 의해 17 murine 바이러스 전염성 ectoparasites, 기생충 endoparasites 및 항체 무료로 확인됩니다. 마우스는 실험실 동물의 관리 및 사용에 대한 안내에 따라 툴란 대학의 AAALAC - 공인 동물 시설에 보관되어있다. 1 </li><li> 특히, 마우스는 나무 침구 개별적으로 통풍 폴리 카보 네이트 cageswith의 microisolation 필터 정상에 보관되어 그룹 (4-5 마우스 / 케이지)입니다. 그들은 광고 libitum pelleted 쥐 음식과 수돗물을 제공하고 있습니다. </li><li> 생쥐는 칠일은 연구의 시작하기 전에 적응을 사용할 수 있습니다. </li></ol> 2. 셀 <ol><li> murine 전환 세포 암종 세포 라인 MB49은 방광 종양을 생산하는 데 사용됩니다. </li><li> 세포 10% 태아 소 혈청, 100 단위 / ML 페니실린, 100 단위 / ML 스트렙토 마이신과 보충 Dulbecco의 수정 이글의 중간에있는 양식입니다. </li><li> 문화가 37 ° C 유지 및 매체와 CO 2 2 ~ 3 일마다 변경 5 %. </li></ol> 3. 동물 Orthotopic 종양 모델 방광 종양 모델 여기서 설명의 기지는 군터 외에 의해 설명하는 절차를 수정합니다. 2 <ol><li> 마취는 산소 isoflurane 2% (MWI / VetOne, 메리디언, ID, 고양이 # 501017)를 유도하고 0.75-1.25 %에서 관리하고 있습니다. (여러 동물 isoflurane 수준의 증가 할거 것이되는, 동시에 처리하실 수 있습니다.) 각막 건조를 방지하기 위해, 눈이 인공 눈물 안과 연고와 윤활 있습니다. 체온은 종이 드레이프 아래 온수 패드로 유지됩니다. </li><li> 허리의 모피는 금속 접지 플레이트와 접촉을 추진 잘린이며, 면도 지역 electroconduction 젤로 코팅됩니다. </li><li> 각각의 마우스는 다음 개별적으로 금속 접지 판에 지느러미 recumbancy에 배치되고 방광은 transurethrally, 4분의 3 인치 이상 - 더 - 바늘 IV 카테터가 제거 탐침과 윤활 24 게이지를 사용하여 catheterized 있습니다. </li><li> 방광은 소변의 탈진 상태이며, 백금 와이어는 카테터의 각 끝에 노출된 전선을 떠나 방광에 카테터를 통해 삽입됩니다 - 카테 테르의 lumenal 끝에 약 2mm. 와이어는 백금의 산화로 인해 소재의 저항 이루어집니다. </li><li> 2.5 W 전류는 다음 응고로 설정 electrocautery 장치와 와이어의 외관 부분을 만져 약 0.5 초에 대한 방광에 적용됩니다. </li><li> 와이어는 다음 방광에서 두 번째 위치와 다른 적용 2.5 W 요금으로 이동됩니다. </li><li> 와이어가 제거하고, / ML은 1ml 주사기를 통해 방광에 카테터를 통해 얻은 것입니다 1x10 6 세포의 농도에서 세포 MB49 100 μl입니다. </li><li> 주사기는 세포 suspensions의 유출을 방지하기 위해 카테터에 연결된, 그리고 catheterized 생쥐는 세포가 화상 사이트에 연결할 수 있도록 90 분 정도 마취 유지됩니다 남아 있습니다. </li><li> 멸균 생리의 정상 100-200 μl는 복구하기 전에 각 마우스 IP 주사로 관리합니다. </li><li> 잠복기의 끝에 카테터가 제거되고 righting의 반사 신경은 생쥐가 자신의 집에서 연습장에 반환되는 시점에서 돌아올 때까지 동물은 온수 패드에 복구할 수 있습니다. </li></ol> 4. 초음파 <ol><li> 종양은 생쥐는 종양 성장을 모니터링하는 방광의 초음파 검사를 받아야하기 전에 3~5일에 대한 성장을 사용할 수 있습니다. </li><li> 위에서 설명한 초기 초음파 시험, 동물 anesthetized 있습니다. 생쥐는 지느러미 recumbancy에 배치하고 있으며, 복부 피부는 모피를 클리핑하여 준비가되어 있습니다. </li><li> 24 게이지 IV 카테터가 방광에 transurethrally 삽입됩니다. 이 시점에서, 방광은 탈진 상태와 소변 추가 분석을 위해 수집하실 수 있습니다. </li><li> 무균 정상 생리를 100 μl은 초음파에서 방광의 가시성을 향상을 위해 카테터를 통해 방광으로 얻은 것입니다. </li><li> 초음파 전도 젤은 복부에 적용되며, 초음파 검사는 방광 벽 및 루멘을 평가하기 위해 수행됩니다. </li></ol> 5. 대표 결과 : 종양 조직은 innoculation 후 3-5일 표시됩니다. 일 5 시까지 - 우리의 경험에서는, 12 마우스의 그룹에 더 정기적으로 50 %가 (1.0 μl, V = π / 6 · L ° W · H를 사용하여 0.5) 방광 내에 감지 조직 질량 있습니다. 메가바이트 49 세포를 사용하는 경우 종양 성장을 계속하고 쥐가 치료 가면 안락사는 24 일 필요로합니다. 쥐가 기면 또는 cachectic 될 때 안락사를 권장합니다, 쓸 모피, hunching, 비 응답 행동하거나 그들의 초기 체중의 20 %를 잃게됩니다. &lt;를 표시/ P&gt; 종양은 빠르게 성장하고 개발 종양의 세포가 빨리 angiogenic 혈액 공급에 의해 서비스를 수있는 것보다 분아 따위에 의해 번식하기 때문에 그들이 표면상 200 MG 이상 3의 질량에 도달 후 괴사성 센터됩니다. 하나는 간접적으로 아마도 개발 종양 내에 급속한 angiogenesis 관련되어 hematurea의 수준을 통해 종양 진행을 모니터할 수 있습니다. 그것은 일관되게 치료 종양 베어링 생쥐의 소변은 14 일 혈액을 포함 있다고 언급했다. 적혈구의 농도는이 생쥐에서 시간이 지남에 따라 증가 등장하고, 소변의 볼륨이 감소 수집했습니다. 50 MG 초과 종양은 또한 일반적으로 십사일 게시물 innoculation에 의해 상대적으로 쉽게 palpated 수 있습니다.

<ol>
	<li>Institute of Laboratory Animal Resources. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals. , (1996).</li><li>Gunther, J. H., Jurczok, A., Wulf, T., Brandau, S., Deinert, I., Jocham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+syngeneic+orthotopic+murine+bladder+cancer+(MB49).">Optimizing syngeneic orthotopic murine bladder cancer (MB49).</a> Cancer Res. 59, 2834-2837 (1999).</li><li>Zhang, X., Atala, A., Godbey, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression-targeted+gene+therapy+for+the+treatment+of+transitional+cell+carcinoma.">Expression-targeted gene therapy for the treatment of transitional cell carcinoma.</a> Cancer Gene Ther. 15, 543-552 (2008).</li></ol>

동물 실험 툴란 대학의 기관 관리 및 사용위원회에 의해 명시된 지침과 규정에 따라 수행되었다.

서로 다른 결과가 예상해야하지만, 여성의 생쥐에 orthotopic 방광 종양 수립이 법으로서 방법, MB49 이외의 셀 유형에도 적용할 수 있습니다. 덜 공격적인 종양 세포를 사용하는 경우 (새로운 최적을 수립 0.5 W 단위 사용) 약간 와트 증가하면 종양 세포 부착에 더받을 수있는 영역을 낼 것이며, 얻은 세포에 대한 더 이상 방광 배양 시간은 큰 금액을 낼 것입니다 화상 사이트 (게시되지 않은 데이터)에 ?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>C57Bl/6J mice</td>
<td>&nbsp;</td>
<td>Jackson Laboratories</td>
<td>000664</td>
<td>3-5-wk-old</td>
</tr>
<tr>
<td>Polycarbonate cages and ventilated rack</td>
<td>&nbsp;</td>
<td>Lab Products</td>
<td>Super Mouse 750</td>
<td>327mm x 190mm x 143mm</td>
</tr>
<tr>
<td>Hardwood Bedding</td>
<td>&nbsp;</td>
<td>Harlan Teklad</td>
<td>Laboratory &#x2013;grade SaniChips</td>
<td>&nbsp;</td>
<tr>
<td>Rodent diet</td>
<td>&nbsp;</td>
<td>Harlan Teklad</td>
<td>Rodent Diet 2019</td>
<td>&nbsp;</td>
<tr>
<td>IV Catheter</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>009231</td>
<td>24 G x &frac34; in. </td>
</tr>
<tr>
<td>Electrocautery unit</td>
<td>&nbsp;</td>
<td>Bovie Medical Corporation</td>
<td>Aaron 900</td>
<td>&nbsp;</td>
<tr>
<td>Ultrasound unit</td>
<td>&nbsp;</td>
<td>Sonosite</td>
<td>180 Plus (Vet)</td>
<td>&nbsp;</td>
<tr>
<td>MB49 cell line</td>
<td>&nbsp;</td>
<td>Anthony Atala, Wake Forest University Baptist Medical Center</td>
<td>N.A.</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21063-029</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Gemini Bio-Products</td>
<td>100106 500A57B</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-163</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

an orthotopic model of murine bladder cancer

이 간단한 절차에서 방광 종양이 catheterization의 사용, 지역 소작하고, 이후 세포 접착을 통해 여성 C57 생쥐에서 설립되었습니다. 그들의 bladders가 transurethrally catheterized과 탈진 후, 동물 다시 요도 또는 방광을 손상시키지 않고 bladders에 백금 와이어의 삽입을 허용하는 catheterized 있습니다. 카테터는 화상 상해를 만들 방광에 전류를 실시 와이어에 대한 절연체 역할을 테플론의 만들어집니다. electrocautery 단위는 세포 레이어를 멀리 레코딩 및 후속 catheterization 통해 방광에 정지에 전달됩니다 암종 세포에 대한 첨부 파일 사이트를 제공하는 전선의 노출 끝에 2.5W 전달하는 데 사용됩니다. 세포는 카테터를 제거하고 bladders가 자연스럽게 배수 수있는 후 90 분 동안 방광에 남아 있습니다. 종양의 발달은 초음파를 통해 모니터링됩니다. 특정주의는 함께 동영상 catheterization 기술로 지급됩니다.

Watch this Scientific Journal Video about An Orthotopic Model of Murine Bladder Cancer at JoVE.com

An Orthotopic Model of Murine Bladder Cancer

방광 종양은 catheterization, 지역 소작하고, 화상 사이트에 암종 세포의 후속 유착을 통해 법으로서 방식으로 여성의 생쥐에 설립되었습니다.

Murine 방광 암의 Orthotopic 모델

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

an-orthotopic-model-of-murine-bladder-cancer

Research

JoVE Journal

Medicine

15.8K 조회수.  Tulane University. 방광 종양은 catheterization, 지역 소작하고, 화상 사이트에 암종 세포의 후속 유착을 통해 법으로서 방식으로 여성의 생쥐에 설립되었습니다.

비디오: An Orthotopic Model of Murine Bladder Cancer

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

구강 편평 세포 암종의 Orthotopic 마우스 모델에서 종양 세포 침략의 다중 광자 이미징

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

연구

의학

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Orthotopic 마우스 뇌종양 모델의 조합 방사선 치료

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

Orthotopic 폐 이식의 Murine 모델에 Obliterative Bronchiolitis의 개발

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

Orthotopic 방광 종양 모델 및 Intravesical saRNA 트리 트먼트의 평가

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

역 형성 갑상선 암의 동소 마우스 모델

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

유전자 전달 연구 동소 방광암 모델

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

경쟁 손쥐 골수 이식 모델에서 기증자 세포 Engraftment를 확인하려면 수량 실시간 PCR을 사용하여

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

췌장암의 진행 발광 동소 모델

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

인간의 전립선 암 전이의 동소 쥐과 모델

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

쥐과 동 소성 방광 이종 이식의 최소 침습 설립

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...