Department of Surgery, University of California, San Francisco Raymond Shaheen, MD

I. Cell Preparation <ol><li> Darmkrebs-Zellen in Kultur gezüchtet und geerntet werden, wenn subkonfluenten. </li><li> Eine einzelne Zelle Suspension wird in Phosphat-gepufferter Saline und auf Eis gehalten. </li></ol> II. Tumor Vorbereitung <ol><li> Eine Maus mit einem vorher festgelegten subkutane kolorektalen Tumor eingeschläfert. </li><li> Die subkutane Tumor wird entfernt, indem sterile Technik und in 2-3 mm Stücke geteilt </li><li> Der Tumor Stücke sind in Phosphat gehalten Kochsalzlösung auf Eis. </li></ol> III. Maus Vorbereitung Hinweis: In unserem Labor verwenden wir eingeatmet Isofluran, um die Maus zu betäuben, alternativ kann man Injektionsnarkosemittel verwenden, um denselben Effekt zu erzielen <ol><li> Die Narkosetiefe wird anhand Zehen klemmen. Es sollte keine zurückzutreten Reflex mit Zehen klemmen. </li><li> Antibiotika können an dieser Stelle gegeben werden. </li><li> Die narkotisierten Maus, die vorher rasiert wurde, richtig positioniert ist. </li><li> Der Bauch ist mit einem betadine Lösung vorbereitet. </li><li> Die Bauch-und OP-Gebiet sind in einer sterilen Mode drapiert. </li></ol> IV. Laparotomie <ol><li> Eine kleine Nick ist in die Haut gemacht </li><li> Die Bauchwandmuskulatur begriffen und hob </li><li> Die Bauchhöhle wird eingegeben und ein einziges Blatt der Schere wird verwendet, um die intra-abdominale Inhalte wegschieben </li><li> Der Schnitt wird um 2-3 cm verlängert </li></ol> V. Exposure des Blinddarms <ol><li> Der Blinddarm mit seinen blind endende Tasche identifiziert und exteriorisierten </li><li> Der Blinddarm ist vom Rest der Maus über einen pre-cut, sterile Gaze isoliert </li><li> Warm Kochsalzlösung wird verwendet, um den Blinddarm feucht zu halten </li></ol> VI. Injektion von Zellen in den Blinddarm Wand <ol><li> A 27 G oder feiner Nadel wird benutzt, um eine 50 ul Volumen von Zellen in den Blinddarm Wand spritzen </li><li> Die Nadel wird entfernt </li><li> Die Einstichstelle wird überprüft, um sicherzustellen, keine Leckage </li><li> Der Blinddarm ist es, die Bauchhöhle zurück </li></ol> VII. Die Transplantation von Tumor auf den Blinddarm Hinweis: Zusätzlich zu injizieren Zellen in den Blinddarm Wand, ist ein alternativer Ansatz zur Tumor auf den Blinddarm Transplantation <ol><li> Eine Figur von 8 Stich ist auf den Blinddarm mit 6-0 oder 7-0 große Naht gelegt </li><li> Der Blinddarm Wand ist leicht beschädigt </li><li> Dann wird der Tumor Stück positioniert </li><li> Der Stich ist festgebunden </li><li> Der Blinddarm ist es, die Bauchhöhle zurück </li></ol> VIII. Maus Bauchwand Closure and Recovery <ol><li> Die Maus Bauchdecke geschlossen ist mit drei Stichen unterbrochen mit 3-0 oder 4-0 große Naht </li><li> Alternativ kann man mit einem einfachen Laufstich </li><li> Postoperative Schmerzmittel und ein Flüssigkeitsbolus kann an dieser Stelle gegeben werden </li><li> Die Maus ist erlaubt, aus der Narkose zu erholen. </li></ol> Hinweis: Bei Inhalationsanästhetika dies in der Regel dauert 30 Sekunden IX. Ergebnisse - Primärtumor und Lebermetastasen <img src="/files/ftp_upload/old/484_2008_2_11_8/A.png" alt="" /><img src="/files/ftp_upload/old/484_2008_2_11_9/B.png" alt="" /><img src="/files/ftp_upload/old/484_2008_2_11_10/C.png" alt="" /> Primary Tumor - in situ gezeigt (A) und mit dem Nachweis der Gefäßneubildung (B), auf H &amp; E-Färbung, sind Tumoren lokal invasive (C) <img src="/files/ftp_upload/old/484_2008_2_11_11/D.png" alt="" /> Lebermetastasen - gezeigt, ex vivo (D)

<ol>
	<li>Heijstek, M. W., Kraneburg, O., Rinkes Borel, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+colorectal+cancer+and+liver+metastases.">Mouse models of colorectal cancer and liver metastases.</a> Dig Surg. 22, 16-25 (2005).</li><li>Kobaek-Larsen, M., Thorup, I., Diederichsen, A., Fenger, C., Hoitinga, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+colorectal+cancer+and+its+metastases+in+rodent+models:+comparative+aspects+with+those+in+humans.">Review of colorectal cancer and its metastases in rodent models: comparative aspects with those in humans.</a> Comp Med. 50, 16-26 (2000).</li><li>Wilmanns, C., Fan, D., O&apos;Brian, C. A., Bucana, C. D., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+and+ectopic+organ+environments+differentially+influence+the+sensitivity+of+murine+colon+carcinoma+cells+to+doxorubicin+and+5-fluorouracil.">Orthotopic and ectopic organ environments differentially influence the sensitivity of murine colon carcinoma cells to doxorubicin and 5-fluorouracil.</a> Int J Cancer. 52, 98-104 (1992).</li><li>Kashtan, H., Rabau, M., Mullen, J. B., Wong, A. H. C., Roder, J. C., Shiptz, B., Stern, H. S., Gallinger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-rectal+injection+of+tumor+cells:+a+novel+animal+model+of+rectal+cancer.">Intra-rectal injection of tumor cells: a novel animal model of rectal cancer.</a> Surg Oncol. 1, 251-256 (1992).</li></ol>

Obwohl Maus subkutanen Tumor-Modellen leicht festzustellen und zu überwachen sind, ist es klar, dass dieses Modell nicht replizieren kann die ursprüngliche anatomische Stelle von Darmkrebs. Aufgrund der Differenz in Mikroumgebung haben kolorektalen Krebszellen wachsen unter der Haut gezeigt, ihren Phänotyp zu ändern und fast immer keine Fortschritte und metastasieren 1,2. In der Tat können Ansprechen des Tumors auf die Therapie erheblich variieren je nachdem, ob Krebszellen in einer Eileiterschwangerschaft (subkutan) versus ...

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		<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>Cell culture media and components</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>will vary depending on cell line used</td>
</tr>
<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>BioWhitaker</td>
<td>17-512F</td>
<td>also sold by a number of other vendors</td>
</tr>
<tr>
<td>15 mL centrifuge tubes, polypropylene</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430790</td>
<td>&nbsp;</td>
<tr>
<td>6 well plates with lid</td>
<td>&nbsp;</td>
<td>Becton Dickinson Labware</td>
<td>353046</td>
<td>&nbsp;</td>
<tr>
<td>Iris forceps, straight, serrated tips</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>15914</td>
<td>best to have a pair of these instruments</td>
</tr>
<tr>
<td>Iris dissecting scissors, straight, sharp</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>14393</td>
<td>&nbsp;</td>
<tr>
<td>Needle Holder (regular)</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>14109</td>
<td>&nbsp;</td>
<tr>
<td>Needle Holder (Castroviejo)</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>14137</td>
<td>&nbsp;</td>
<tr>
<td>Cidex Plus</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>7364</td>
<td>instruments should be autoclaved periodically, but may also be sterilized by soaking in Cidex</td>
</tr>
<tr>
<td>6-0 or 7-0 suture</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for cecum; courtesy of Department of Surgery, UCSF</td>
</tr>
<tr>
<td>3-0 or 4-0 suture</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for abd wall; courtesy of Department of Surgery, UCSF</td>
</tr>
<tr>
<td>Anesthetic machine / Isoflurane</td>
<td>&nbsp;</td>
<td>J.A. Baulch and Associates</td>
<td>3206</td>
<td>also sold by a number of other vendors</td>
</tr>
<tr>
<td>Heating Pad, disposable, 6" x 6"</td>
<td>&nbsp;</td>
<td>Prism Technologies</td>
<td>20419</td>
<td>&nbsp;</td>
<tr>
<td>Tape, orange, 13 mm x 13 m</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15901F</td>
<td>for restraining anesthetized mouse</td>
</tr>
<tr>
<td>Pro-Cord/Cordless Trimmer</td>
<td>&nbsp;</td>
<td>Oster</td>
<td>78997-010</td>
<td>for shaving mouse fur</td>
</tr>
<tr>
<td>Betadine</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>19-027132</td>
<td>may also be purchased at any medical supply store</td>
</tr>
<tr>
<td>2 x 2 gauze, 3-ply</td>
<td>&nbsp;</td>
<td>Johnson and Johnson</td>
<td>7635</td>
<td>may also be purchased at any medical supply store</td>
</tr>
<tr>
<td>Sterile Field, Barrier, 16" x 29"</td>
<td>&nbsp;</td>
<td>Johnson and Johnson</td>
<td>0905</td>
<td>may also be purchased at any medical supply store</td>
</tr>
<tr>
<td>Cotton tipped applicators, 6"</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>14-960-3Q</td>
<td>may also be purchased at any medical supply store</td>
</tr>
<tr>
<td>Syringe, 10 cc, Luer lock tip</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309604</td>
<td>&nbsp;</td>
<tr>
<td>Syringe, 1 cc, tuberculin, slip tip</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309602</td>
<td>&nbsp;</td>
<tr>
<td>27 1/2 G needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>305109</td>
<td>&nbsp;</td>
<tr>
<td>30 G needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>305106</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
					</div>

orthotopic mouse model of colorectal cancer

Die traditionellen subkutanen Tumor-Modell ist weniger als ideal für das Studium des kolorektalen Karzinoms. Orthotopen Mausmodell des kolorektalen Karzinoms, die Krebszellen wachsen in ihrem natürlichen Standort-Funktion, replizieren menschlichen Krankheiten mit hoher Wiedergabetreue. Zwei Techniken können verwendet werden, um dieses Modell zu etablieren. Beide Techniken sind ähnlich und erfordern Maus Anästhesie und Laparotomie für die Exposition des Blinddarms. Eine besteht darin, Injektion eines kolorektalen Karzinoms Zellsuspension in den Blinddarm Wand. Krebszellen werden zuerst in Kultur angebaut, geerntet, wenn subkonfluenten und hergestellt, wie eine einzelne Zelle Suspension. Ein kleines Volumen der Zellen wird langsam, um ein Auslaufen zu vermeiden injiziert. Die andere Technik beinhaltet die Transplantation von einem Stück des subkutanen Tumor auf den Blinddarm. Eine Maus mit einem vorher festgelegten subkutane kolorektalen Tumor eingeschläfert und der Tumor wird entfernt, indem sterile Technik. Der Tumor Stück ist in kleine Stücke für die Transplantation zu einem anderen Maus unterteilt. Vor der Transplantation wird der Blinddarm Wand leicht beschädigt, um Tumor-Zell-Infiltration zu erleichtern. Die Zeit bis zur Entwicklung von Primärtumoren und Lebermetastasen hängt von der Technik-, Zell-Linie, und die Maus Arten verwendet. Diese orthotopen Mausmodell ist nützlich für die Untersuchung der natürlichen Entwicklung von Darmkrebs und Erprobung neuer Therapeutika gegen den Darmkrebs.

Watch this Scientific Journal Video about Orthotopic Mouse Model of Colorectal Cancer at JoVE.com

Orthotopic Mouse Model of Colorectal Cancer

Zwei Techniken können verwendet werden, um dieses Modell zu etablieren werden: Injektion einer Krebszelle Suspension in den Blinddarm Wand oder Transplantation von einem Stück des subkutanen Tumor auf den Blinddarm. Dieses Modell ist nützlich für die Untersuchung der natürlichen Entwicklung von Darmkrebs und Erprobung neuer Therapeutika gegen den Darmkrebs.

Orthotopen Mausmodell der Darmkrebs-

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

orthotopic-mouse-model-of-colorectal-cancer

Research

JoVE Journal

Biology

44.7K Aufrufe.  University of California, San Francisco - UCSF. Zwei Techniken können verwendet werden, um dieses Modell zu etablieren werden: Injektion einer Krebszelle Suspension in den Blinddarm Wand oder Transplantation von einem Stück des subkutanen Tumor auf den Blinddarm. Dieses Modell ist nützlich für die Untersuchung der natürlichen Entwicklung von Darmkrebs und Erprobung neuer Therapeutika gegen den Darmkrebs.

Serie: Orthotopic Mouse Model of Colorectal Cancer

Chronic Salmonella Infected Mouse Model

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo. Mice with Salmonella persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.

Chronic Salmonella Infected Mouse Model

Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.

Chronisch Salmonellen Infected Maus-Modell

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

Forschung

Biologie

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Ableitung der Maus Trophoblast Stammzellen aus Blastozysten

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

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.

In-vivo-Imaging und therapeutische Behandlungen in einem orthotopen Mausmodell des Eierstockkrebs

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

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Durchflusszytometrie Reinigung der Maus meiotischen Zellen

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

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.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

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

En Face Preparation of Mouse Blood Vessels

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the three-dimensional tissue morphology is from such sections. In addition, the sections of tissues examined may not contain the region within the tissue that is necessary for the purpose of the ongoing study. This latter limitation hinders histopathological studies of blood vessels since vascular lesions develop in a focalized manner. This requires a method that enables us to survey a wide area of the blood vessel wall, from its surface to deeper regions. A whole mount en face preparation of blood vessels fulfills this requirement. In this article, we will demonstrate how to make en face preparations of the mouse aorta and carotid artery and to immunofluorescently stain them for confocal microscopy and other types of fluorescence-based imaging.

En Face Preparation of Mouse Blood Vessels

A procedure for making en face preparations of the mouse carotid artery and aorta is described. Such preparations, when immunofluorescently stained with specific antibodies, enable us to study localization of proteins and identification of cell types within the entire vascular wall by confocal microscopy.

En Face Vorbereitung der Maus Blutgefäße

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-innere Zelle Masse Injektion von embryonalen Stammzellen führt zu höheren Chimerism Raten

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Ein Maus-Modell der teilweisen Darmverschluss

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Purification of Platelets from Mouse Blood

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. We describe here purification of platelets from mouse blood using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 &#176;C. The recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

Reinigung von Plateletten aus dem Mausblut

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.