이 작품은 CIRM 보조금 # TR-1250, RT1-01108 및 CL1-00502에 의해 재정 지원되었다.

참고 : 모든 동물의 절차 IACUC 또는 동급의 승인을 받아야합니다. 모든 수술 장비는 수술 전에 소독을해야합니다. 멸균 장갑, 커튼과 거즈를 사용해야합니다. 1. 수술에 대한 사전 준비 <ol><li> 6주 오래된 뮤스 Musculus CbySmn.CB17-Prkdc SCID / J 수컷 마우스 또는 마우스의 다른 immunocompromised 긴장을 구합니다. </li><li> 모든 수술 도구, 장갑, 그리고 거즈를 소독. </li><li> accutase로 주입되는 hPSCs을 떼어 놓다. </li><li> 세포를 세고 matrigel은 DMEM/F-12에서 1:1 희석에서 20-30 μL 당 1,000,000 세포를 resuspend. 완료하기 전까지 얼음 세포를 유지. </li><li> 종종 Trypan 블루가 주입되는 실험을 통해 실행을 할 때 유용합니다. 이것은 모든 세포가 낭비되기 전에 잠재적인 문제를 식별하는 데 도움이 될 것입니다. </li><li> 동식물 사육장에서 해당 기관의 승인 절차에 따라 마우스를 마취. 우리의 실험에서, 우리는 anesth를 사용isoflurane과 esia 기계. 마우스는 처음 1L/min 산소와 3-4% isoflurane으로 유도 챔버에 넣어되었습니다. 일단 anesthetized, 1L/min 산소와 2-3% isoflurane과 코 콘이 사용되었습니다. </li><li> 가위로 복부를 면도하고, 복부의 중앙에서 시작하여 바깥쪽으로 시계 방향으로 작동, 마우스 복부의 앞부분은 벽을 청소. 처음 사용할 때 Povidone-요오드 용액 후 70 % 에탄올로 씻는다. 면봉 때마다 변화, 3 번 반복합니다. 조직 배양 또는 해부 후드 내부 동물 보온하는 가열 패드로 동물을 전송합니다. </li><li> 단지 공동 고관절의 수준 아래 무균 수술 가위로 피부와 복막을 통해 1cm 세로 절개를합니다. </li><li> 집게와 함께 복막을 누른 상태에서 다른 멸균 forcept과 오른쪽 엉덩이쪽으로 내려 도달 첨부 testes와 함께 흰색 지방 조직을 당겨. </li><li> 멸균 거즈에 testes를 놓습니다. </li><li> 투베르쿨린 또는 해밀턴 (1cc) 주사기 WI를 채우hPSCs가 주입되는 전 ... 당신 같은 WA09 (또한 H9라고도 함)로 pluripotent 알고있는 컨트롤 hPSC 라인을 포함하는 것이 좋습니다합니다. 이렇게 당신은 당신의 주입이나 수술 결함 여부를 확인하는 데 사용할 수있는 긍정적인 제어할 수 있습니다. </li><li> 천천히 고환 캡슐이 팽창하기 시작하면 중지, 주요 혈관에서 떨어져 testis 캡슐의 중심에 hPSCs (20-30 μL)을 주입. </li><li> 세포의 역류를 방지하기 위해 천천히 주사 바늘을 제거합니다. </li><li> 집게를 사용하여 복부를 원래 위치로 testes과 지방 조직을 다시 전송합니다. </li><li> 2 ~ 3 reabsorbable 봉합과 복막을 닫고 autoclips으로 피부를 닫습니다. </li><li> 그것이 복구로 1-2 일 동안 하루에 두 번 수술 후 진통제의 일부 형태 (귀하의 동식물 사육장에서 허용되는 참조) 주어진 때까지 마우스는 따뜻한 보관해야합니다. 로 마취도 진통도는 종양 발달을 방해합니다. </li><li> 일을 모니터링6-12 주 동안 종양의 성장을위한 전자 동물. 아주 드물게, 종양은 사전에 6주 주입 후의 크기가 5mm로 성장할 수 없습니다, 따라서 그것은 동물을 모니터링하는 것이 중요합니다. 이 경우 동물 euthanized과 종양 처리하고 정상적으로 분석되어야한다. </li><li> 종양이 만져서 알 수있는이고 크기가 약 5mm 도달되면, 마우스를 마취 및 접수 동물 프로토콜 절차에 따라 그것을 희생. </li><li> 이에 따라 종양 및 문서 제거 - 사진, 측정 크기, 무게. </li><li> 작은 조각으로 종양을 잘라 4 % paraformaldehyde 용액에 고정시킨다. 샘플까지 4 % paraformaldehyde의 상점은 sectioning, 염색법 및 분석을 위해 병리학자로 전송됩니다. </li></ol> 2. 대표 검색 결과 : 이 프로토콜이 완료되면 같이 설명하고 주입 세포 라인 pluripotent이며, 만져서 알 수있는, 시각적으로 명백한, 종양이 가장 12 주 이내에 구성해야합니다. WA09 같은 설립 hPSC 라인을 위해, 우리는 일반적으로 볼 수육주 이내에 종양. iPSC 라인의 경우는 8-10주 내에 종양을보고 드문되지 않습니다. 그것은 절차가 올바르게 수행되었는지 확인하기 위해서는, 긍정적인 컨트롤로 pluripotent 것으로 알려져 라인 쥐 주입하는 것이 매우 중요합니다. 종양은 대개 매우 이질적인 모양과 많은 첨부된 cysts (그림 1)가. 병리학자에 의한 종양 샘플의 분석 세 배아 레이어 (그림 2)에서 차별화된 조직을 표시해야합니다. <img alt="그림 1" src="/files/ftp_upload/3177/3177fig1.jpg" /> 그림 1. testis 캡슐의 일반 hPSC 파생 teratoma. 설명된 프로토콜에 따라 백만 WA09 세포 면역 훼손 마우스 testis 캡슐에 주입했다. 6 주 후 teratoma가 관찰되었다. ) Teratoma는 마우스에서 가져온. B) teratoma의 사진을 닫습니다. 이질과 낭종 구조를합니다. <img alt="그림 2" srC = &quot;/ files/ftp_upload/3177/3177fig2.jpg&quot;/&gt; 그림 2. Hematoxylin과 Eosin teratoma의 스테인드 섹션은 각 세균 층의 조직을 보여줍니다. 고정 따라 teratoma은 Hematoxylin과 Eosin으로 sectioned 및 스테인드했습니다. 병리학자에 의한 분석 3 배아 레이어 각각의 세포의 존재를 공개했다.

<ol>
	<li>Bradley, A., Evans, M., Kaufman, M. H., Robertson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+germ-line+chimaeras+from+embryo-derived+teratocarcinoma+cell+lines.">Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines.</a> Nature. 309, 255-255 (1984).</li><li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154 (1981).</li><li>Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+pluripotent+cell+line+from+early+mouse+embryos+cultured+in+medium+conditioned+by+teratocarcinoma+stem+cells.">Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.</a> Proc. Natl. Acad. Sci. U. S. A. 78, 7634-7634 (1981).</li><li>Downing, G. J., Battey, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+assessment+of+the+first+20+years+of+research+using+mouse+embryonic+stem+cell+lines.">Technical assessment of the first 20 years of research using mouse embryonic stem cell lines.</a> Stem Cells. 22, 1168-1168 (2004).</li><li>Thomson, 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=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1145 (1998).</li><li>Reubinoff, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+from+human+blastocysts:+somatic+differentiation+in+vitro.">Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.</a> Nat Biotechnol. 18, (2000).</li><li>Muller, F. J., Goldmann, J., Loser, P., Loring, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+call+to+standardize+teratoma+assays+used+to+define+human+pluripotent+cell+lines.">A call to standardize teratoma assays used to define human pluripotent cell lines.</a> Cell Stem Cell. 6, 412-412 (2010).</li><li>Brivanlou, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells.+Setting+standards+for+human+embryonic+stem+cells.">Stem cells. Setting standards for human embryonic stem cells.</a> Science. 300, 913-913 (2003).</li><li>Lensch, M. W., Schlaeger, T. M., Zon, L. I., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratoma+formation+assays+with+human+embryonic+stem+cells:+a+rationale+for+one+type+of+human-animal+chimera.">Teratoma formation assays with human embryonic stem cells: a rationale for one type of human-animal chimera.</a> Cell Stem Cell. 1, 253-25 (2007).</li><li>Gertow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organized+development+from+human+embryonic+stem+cells+after+injection+into+immunodeficient+mice.">Organized development from human embryonic stem cells after injection into immunodeficient mice.</a> Stem. Cells. Dev. 13, 421-421 (2004).</li><li>Gertow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+embryonic+stem+cell-derived+teratomas+for+the+assessment+of+pluripotency.">Isolation of human embryonic stem cell-derived teratomas for the assessment of pluripotency.</a> Curr Protoc Stem Cell Biol. Chapter 1, Unit1B 4-Unit1B 4 (2007).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

여기에 제시된 방법은 testis 캡슐에 hPSCs에서 기형 종을 발생시키는 매우 안정적이고 직접적인 수단을 제공합니다. 이 기법의 몇 가지 중요한 매개 변수가 있습니다. 특히, 그것은 통제와 같은 pluripotent 것으로 알려져 있습니다 hPSC 세포 라인을 주입하는 것이 중요합니다. 다른 중요한 파라미터는 분사와 종양 관찰 사이의 시간 간격이 포함됩니다. WA09 같은 세포 라인의 경우, 기형 종은 6~8주에서 관찰되어야한다. 새로운 iPSC 라인의 경우 우리는 자주 10주이 필요다는 것을 찾을 수 있습니다. 또 다른 우려는 주입된 세포의 숫자입니다. 우리가 백만 세포를 주입했지만 검정은 쉽게 적은 수의 세포로 할 수 있습니다. 또한, 사출 매체는 중요하다. 우리는 PBS 또는 DMEM/F-12 반대로 세포, matrigel과 DMEM/F-12의 1:1 혼합물에 주입 때 우리가 최상의 결과를 얻을 것을 발견했습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> Accutase </td><td> Invitrogen </td><td> A1110501 </td></tr><tr><td> DMEM/F-12 </td><td> Invitrogen </td><td> 113300-032 </td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354,277 </td></tr></tbody></table>

teratoma generation in the testis capsule

Pluripotent 줄기 세포는 (PSCs)들은 세 배아 레이어의 세포로 구별 수있는 독특한 특성이 있습니다. 이것은 그들 여러 질병의 치료를 위해 잠재적으로 유용한 도구가 있습니다. 유도된 pluripotent 줄기 세포 (iPSCs)와 인간 배아 줄기 세포와 지속적인 연구의 출현과 함께 (hESCs) 특정 세포 라인 pluripotent임을 증명할 수있는 assays를위한 필요가있다. Germline 전송 마우스 배아의 줄기 세포 (mESC) 라인 1,2,3의 pluripotence를 시연을위한 황금 표준되었습니다. 이 분석 사용하여 연구자들은 mESC 라인이 배아 세포 네를 포함한 배아에있는 모든 세포 유형을 만들 수있다는 것을 보여줄 수있다. 인간 ESCs가 germline 전송에 대한 검사를받을 수 없기 때문에 인간의 ESC 라인 5,6의 생성을 통해 이러한 세포의 pluripotence 증명할 수있는 적절한 분석은 불분명했습니다. 대리로서 teratoma 분석은 현재 pluripot을 설명하는 데 사용됩니다인간 pluripotent 줄기 세포 ency (hPSCs) 7, 8, 9. 이 분석은 최근 주목 받고왔다하고 새로운 기술을 적극적으로 모색되고 있지만, teratoma 분석은 현재 금 본위 7입니다. 본 분석에서는 문제의 세포는 면역 손상 마우스에 주입된다. 세포 pluripotent 경우 teratoma 결국 개발되고 종양의 섹션은 3 개의 배아 층 10 일부터 조직을 보여줍니다. teratoma 분석에서는 hPSCs는 마우스의 다른 분야로 주입 할 수 있습니다. 또는 다리 중 subcutaneously 또는 intramuscularly 11로, 가장 일반적인 사출 사이트 testis 캡슐, 신장 캡슐, 간 등이 있습니다. 여기에서 우리는 종양 성장을위한 사이트로 testis 캡슐을 사용 hPSCs에서 기형종의 생​​성을위한 강력한 프로토콜을 설명합니다.

Watch this Scientific Journal Video about Teratoma Generation in the Testis Capsule at JoVE.com

Teratoma Generation in the Testis Capsule

인간 pluripotent 줄기 세포 (hPSCs)은 다양한 질병을 치료하기 위해 무수히 많은 가능성이 있습니다. 이러한 세포의 유틸리티들은 신체의 모든 세포 유형으로 구별 수있다는 사실에있다. 여기 hPSCs의 pluripotence을 설명하는 데 사용됩니다 teratoma 분석을 설명합니다.

Testis 캡슐의 Teratoma 생성

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

teratoma-generation-in-the-testis-capsule

Research

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Medicine

20.6K 조회수.  Scripps Research Institute. 인간 pluripotent 줄기 세포 (hPSCs)은 다양한 질병을 치료하기 위해 무수히 많은 가능성이 있습니다. 이러한 세포의 유틸리티들은 신체의 모든 세포 유형으로 구별 수있다는 사실에있다. 여기 hPSCs의 pluripotence을 설명하는 데 사용됩니다 teratoma 분석을 설명합니다.

비디오: Teratoma Generation in the Testis Capsule

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

IMAP 테스트 배터리 : 듣기의 감각을 만들기

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

연구

의학

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

같은 자폐증과 정신 분열증과 같은 일반적인 장애에 드 노보 변이를 식별하는 전략

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

마우스 Aortocaval 누관의 기술적 측면

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

전립선 발암 및 양성 전립선 비대증의 평가를위한 신장 캡슐 Xenografting 및 피하 펠렛 주입

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

생성 및 마우스의 동맥 병변의 3 차원 정량 광학 프로젝션 단층 촬영을 사용하여

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

차세대 시퀀싱에 대한 수집 및 타액 DNA의 추출

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

에서 재조합 효소 중재 카세트 교환에 의해 재조합 인간 다 능성 줄기 세포의 신속하고 효율적인 생성<em&gt; AAVS1</em&gt; 로커스

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Drug Repurposing Hypothesis Generation Using the &quot;RE:fine Drugs&quot; System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

은 &quot;RE를 : 좋은 약&quot;을 사용하여 의약품 용도 변경 가설 발전 시스템

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology. Testicular organoids allow for the study of cell-cell interactions, tissue development, and the germ cell niche microenvironment and facilitate high throughput drug and toxicity screening. A method is needed to reliably and reproducibly generate testicular organoids with testis specific tissue architecture. The microwell culture system contains a dense array of pyramid-shaped microwells. Testicular cells derived from pre-pubertal testes are centrifuged into these microwells and cultured to generate testicular organoids with testis-specific tissue architecture and cell associations. Thousands of homogeneous organoids can be generated via this process. The protocol reported here will be of broad interest to researchers studying male reproduction.

Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system.

마이크로웰 배양을 이용한 고환 특이적 구조를 이용한 돼지 고환 오르가노이드 생성

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs ...