우리를 통해 초기 cloacal 마사지, 호세 Noguera를 감사 하 고 제프리 Kittilson 연구소에 대 한 지원. 이 작품을 BJH ND EPSCoR FAR0022429 수상에 의해 지원 되었다. 출판 비용 아낌없이 NDSU 학과 생물 과학을 지원 했다.

이 프로토콜은 척추 동물 주제를 포함 하 고 기관 동물 관리 및 사용 위원회 (IACUC) 노스 다코타 주립 대학에 의해 승인 되었습니다.
1. 정액 passerine 새 cloacal 마사지를 사용 하 여 컬렉션
참고: Cloacal 마사지 재생산으로 활동적인 새만, 효과적인 정액 컬렉션 기법 이지만, 적절 한 조임 상황에서 번 식 시즌 밖에 성공적으로 수행할 수 있습니다. 생식 활동 cloacal 마사지를 사용 하 여 이전 대상 종에 대 한 결정 되어야 합니다. 캡처 시 야생 조류 또는 조류 포로에서 개최에이 기술을 사용할 수 있습니다.
<ol><li>(사육 조건)에 남성 새부터 손바닥 (그림 1)를 만지고 그것의 복 부 측으로 지배적인 손을 비 지배적인 손에 bander의 그립 전송 새.<ol>
<li>새의 머리는 손바닥의 측면 가장자리에 가까운 인지 확인 합니다. 가볍게 머리와 몸을 핑 키, 반지, 그리고 가운데 손가락을 사용 하 여 보호 합니다. 이것은 조류의 환기와 꼬리 노출.</li>
		<li>느슨한 또는 가볍게 손바닥을 사용 하 여 절제 된 새의 다리를 둡니다.</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55324/55324fig1.jpg"> 그림 1 : Cloacal 마사지에 대 한 그립. 새는 복 부 쪽 손바닥과 머리 핑 키 손가락에 의해 보안 감동 지배적인 손에 가볍게 확보 된다. <a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<ol start="2"><li>지배적인 검지와 엄지는 cloaca의 우수한 끝의 양쪽에 놓고 가볍게 함께 ( 그림 2) 꼬리 쪽으로 아주 약간의 압력을 적용 하는 동안 손가락을 꼬집어. cloaca cloacal 점 막 (핑크 인테리어) 노출 evert 것입니다. 참고: 새 하지 않으면 정액, 현재 생산 cloacal 융기 작은 수 고는 cloaca evert 어렵다. 경험, 재생산으로 활성 및 비활성 남성5,11의 cloacas 사이 분화 하기 쉽습니다.</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55324/55324fig2.jpg"> 그림 2 : 손가락 위치와 cloacal 융기. 우수한 끝 (A)을 약간의 압력을 적용 하 여는 cloaca evert. 재생산으로 활동적인 남성 참새목 cloacal 융기는 분명 하 고 회사 (B). <a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<ol start="3"><li>비 지배적인 손을 가진 안정 손, 꼬리의 기지의 등 쪽 측에 검지 손가락의 패드를 놓고 나왔으며 cloaca 바로 아래 엄지손가락의 끝을 놓습니다. 참고: 짧고 부드러운 축소판 cloacal 마사지를 수행 하기 위한 유리한 이며 실수로 부상에서 새를 보호 한다.</li>
	<li>아래는 cloaca 머물고, 중간 압력으로 반복적인 움직임에 cranially 깊은 비 지배적인 엄지 이동. 새는 사정 될 때까지 계속 합니다. 개인에 따라이 일반적으로 1, 몇몇 다스 선 사이의 걸립니다.</li>
	<li>최대한 빨리 사정 하는 새, microhematocrit 튜브에 정액을 수집 합니다. 여러 ejaculates 얻기 위해 cloacal 마사지를 반복 합니다. 참고: Passerine 정액은 밝은 갈색 및 일반적으로 다소 두꺼운 견실 함이 있다. cloaca에서 배출 하는 흰색 또는 어두운 물질 정액, 고 정액 샘플을 오염 시킬 수 있습니다. cloaca에 혈액의 작은 금액의 외관에 혼합 또는 정액 일반적으로 마사지 하는 동안 너무 많은 압력에 의해 발생.</li>
	<li>DNA 추출에 대 한 1 x PBS (pH 7.4)의 20 µ L 가진 microcentrifuge 관에 정액을 놓습니다. 참고: 1 x PBS에 저장 된 정액 인공 수정에 적합 하지 않습니다 하지만 DNA 추출 나중에 사용할 수 있습니다-80 ° c.에 저장 될 때 PBS 실내 온도 될 수 또는 냉장, 있지만 정액 샘플 예금 될 때 액체 상태에 있어야 한다.<ol>
<li>적은 양의 화합물 현미경 (그림 3) 희석된 샘플을 보고 샘플에서 정자의 존재를 확인 합니다.</li>
	</ol>
</li>
	<li>추출까지-80 ° C에 희석된 정액을 저장 합니다.</li>
</ol>2입니다. 새 정액에서 DNA 추출
참고:이 프로토콜 여러 상업 DNA 추출 키트, 테스트 되었습니다 하지만 단지 하나의 DNA 키트12와 함께 사용 하면 성공 했습니다. 단계를 수정 키트 프로토콜에서 표시 됩니다 *.
<ol><li>물에서 dithiothreitol (DTT)의 1 분 솔루션을 준비 하 고는 vortex.*를 사용 하 여 혼합<ol>
<li>약 수 DTT 솔루션 사용 직전까지-20 ° C에서 그것을 저장 하 고. 각 샘플 추출에 필요한 1 M DTT solution.*의 10 µ L</li>
	</ol>
</li>
	<li>20 mM Tris-Cl, pH 8.0;을 포함 하는 버퍼를 준비 20 mM EDTA; 200 mM NaCL; 그리고 4 %SDS. 각 샘플 추출에 필요한이 buffer.*의 90 µ L<ol>
<li>실 온에서 버퍼를 저장 합니다. 그것은 시 켰 던 경우 dissolve.*에 56 ° C에 따뜻한</li>
	</ol>
</li>
	<li>포함 하는 로커 또는 65 흔드는 인큐베이터를 예 열 ° C.*</li>
	<li>바로 시작 하기 전에 추출, 10 µ L DTT 솔루션 10 샘플을 추출 하는 경우 각 샘플을 추출, 예: 900 µ L 버퍼 플러스 100 µ L DTT 솔루션 단계 2.2에서에서 준비 하는 버퍼의 90 µ L 믹스. 이 혼합물 DTT buffer.* 라는 것입니다.</li>
	<li>70 µ L 1 x PBS 전체 희석된 정액 샘플을 추가 하 고는 vortex.*를 사용 하 여 잘 혼합</li>
	<li>100 µ L를 추가 DTT 버퍼 및 10 µ L 성분 K 솔루션 샘플을. 20 초간 소용돌이 다음 품 어 따뜻한 인큐베이터에서 1 시간 로커 또는 shaker.*에 대 한</li>
	<li>200 µ L 버퍼 알 및 200 µ L 추가 신선한 EtOH (100%)와 20 seconds.*에 대 한 소용돌이</li>
	<li>전체 샘플 스핀 열을 전송 하 고 원심 6000 x g. 삭제에서 1 분 컬렉션 튜브.</li>
	<li>새로운 튜브에 열을 놓고 500 µ L 준비 버퍼 AW1를 추가 합니다. 6000 x g. 삭제에서 1 분 컬렉션 튜브 원심.</li>
	<li>새로운 튜브에 열을 놓고 500 µ L 준비 버퍼 AW2 추가 합니다. 6000 x g.에서 1 분간 원심 분리기는 흐름을 통해 장소와 열 튜브에 다시 삭제 합니다.</li>
	<li>원심 20000 x g. 삭제 3 분 컬렉션 튜브 고 1.5 mL microcentrifuge 튜브에는 열을 놓습니다.</li>
	<li>피펫으로 35 AE의 µ L 열 막에 직접 버퍼 및 5 신속성에 대 한 실 온에서 품 어</li>
	<li>원심 1 분 14000 x g. 삭제에서 스핀 열 고-80 ° c.에서 추출한 샘플을 저장</li>
</ol>

<ol>
	<li>Briskie, J. V., Montgomerie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+size+and+sperm+competition+in+birds.">Sperm size and sperm competition in birds.</a> Proc. R. Soc. B. 247, 89-95 (1992).</li><li>Monaghan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres+and+life+histories:+the+long+and+the+short+of+it.">Telomeres and life histories: the long and the short of it.</a> Ann. N.Y. Acad. Sci. 1206, 130-142 (2010).</li><li>Carrell, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+of+the+male+gamete.">Epigenetics of the male gamete.</a> Fertility and Sterility. 97 (2), 267-274 (2012).</li><li>Burrows, W. H., Quinn, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+collection+of+spermatozoa+from+the+domestic+fowl+and+turkey.">The collection of spermatozoa from the domestic fowl and turkey.</a> Poultry Science. 16 (1), 19-24 (1937).</li><li>Wolfson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cloacal+protuberance:+a+means+for+determining+breeding+condition+in+live+male+birds.">The cloacal protuberance: a means for determining breeding condition in live male birds.</a> Bird-Banding. 23 (4), 159-165 (1952).</li><li>Kast, T. L., Ketterson, E. D., Nolan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+ejaculate+quality+in+dark-eyed+juncos+according+to+season,+stage+of+reproduction,+and+testosterone+treatment.">Variation in ejaculate quality in dark-eyed juncos according to season, stage of reproduction, and testosterone treatment.</a> The Auk. 115 (3), 684-693 (1998).</li><li>Helfenstein, F., Podevin, M., Richner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+morphology,+swimming+velocity,+and+longevity+in+the+house+sparrow+Passer+domesticus.">Sperm morphology, swimming velocity, and longevity in the house sparrow Passer domesticus.</a> Behav. Ecol. Sociobiol. 64, 557-565 (2010).</li><li>Purchase, C. F., Earle, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifications+to+the+IMAGEJ+computer+assisted+sperm+analysis+plugin+greatly+improve+efficiency+and+fundamentally+alter+the+scope+of+attainable+data.">Modifications to the IMAGEJ computer assisted sperm analysis plugin greatly improve efficiency and fundamentally alter the scope of attainable data.</a> J. Appl. Icthyol. 28, 1013-1016 (2012).</li><li>Gill, P., Jeffreys, A. J., Werrett, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forensic+application+of+DNA+'fingerprints'.">Forensic application of DNA 'fingerprints'.</a> Nature. 318 (12), 577-579 (1985).</li><li>Criscuolo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+PCR+assay+for+measurement+of+avian+telomeres.">Real-time quantitative PCR assay for measurement of avian telomeres.</a> J. Avian Biol. 40, 342-347 (2009).</li><li>Lovette, I. J., Fitzpatrick, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cornell Lab of Ornithology Handbook of Bird Biology, 3rd Edition. , (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> QIAamp DNA Micro Handbook. , (2014).</li><li>Carter, K. L., Robertson, B. C., Kempenaers, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+differential+DNA+extraction+method+for+sperm+on+the+perivitelline+membrane+of+avian+eggs.">A differential DNA extraction method for sperm on the perivitelline membrane of avian eggs.</a> Mol. Ecol. 9, 2149-2154 (2000).</li><li>Birkhead, T. R., Buchanan, K. L., Devoogd, T. J., Pellatt, E. J., Catchpole, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Song,+sperm+quality+and+testes+asymmetry+in+the+sedge+warbler.">Song, sperm quality and testes asymmetry in the sedge warbler.</a> Anim. Behav. 53, 965-971 (1997).</li><li>L&#252;pold, S., Calhim, S., Immler, S., Birkhead, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+morphology+and+sperm+velocity+in+passerine+birds.">Sperm morphology and sperm velocity in passerine birds.</a> Proc. R. Soc. B. 276, 1175-1181 (2009).</li><li>Samour, J. H., Smith, C. A., Moore, H. D., Markham, 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=Semen+collection+and+spermatozoa+characteristics+in+budgerigars+(Melopsittacus+undulates).">Semen collection and spermatozoa characteristics in budgerigars (Melopsittacus undulates).</a> Vet. Rec. 118, 397-399 (1986).</li><li>Stelzer, G., Crosta, L., B&#252;rkle, M., Krautwalt-Junghanns, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attempted+semen+collection+using+the+massage+technique+and+semen+analysis+in+various+psittacine+species.">Attempted semen collection using the massage technique and semen analysis in various psittacine species.</a> J. Avian Med. Surg. 19 (1), 7-13 (2005).</li></ol>

저자 들은 아무 경쟁 금융 관심사 선언 합니다.

재생산으로 활성 상태에 있는 중소 조류에서 정액을 수집 하 고 조류 정액에서 DNA를 추출 하는 간단 하 고 신뢰할 수 있는 방법을 설명 합니다.
설명된 정액 DNA 추출 프로토콜 편의상 키트에서 수정 했지만 조류 정액에서 사용 하기 위해 정제 된. 정자는 표준 추출 화학 물질9,13, 세포 및 passerines에서 수집 된 정액 샘플은 매우 작은 (?...

새 생물학 연구에서 널리 이용 되 고 정자 수 쉽게 샘플링할 cloacal를 사용 하 여 정자 품질 및 경쟁1, 정자 telomere 역학2, epigenetics3및 유사한 주제를 포함 하는 연구를 위한 이상적인 주제는 마사지. Cloacal 마사지 새4,,56에서 정액을 모으기를 위한 부드러운, 비-침략 적 수동 기술입니다. 반복된 샘플을 쉽게 얻을 수 있습니다 그리고 특별 한 도구가 필요, 필드 또는 실험실에서 수행 하는 간단 하 합니다. Cloacal 마사지 수십 년 동안 사용 되었습니다, 비록 사용할 수 설명 작성에서 배울 하기가 어렵습니다. 이 게시는 시간과 cloacal 마사지 학습에 관련 된 불확실성을 줄일 것입니다.
Cloacal 마사지를 사용 하 여 새 들에서 수집 하는 정액 운동 성 평가4,7 또는8인공 수정 대 한 즉시 사용 하거나 고급 이미징, DNA 추출 등 다른 용도 대 한 동결 수 있습니다. Passerine 새에서 정액 샘플 작은 하지만 밀도가 포장된 정자를 포함. DNA는 정자9의 특수 보호 기능을 극복 하기 위해 수정 편의상 상업 추출 키트를 사용 하 여 추출 됩니다. 추출 후 정자 telomeres 정량10를 사용 하 여 측정 수 있다.

<table border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microhematocrit capillary tubes</td>
			<td style="width:71px;">Fisherbrand</td>
			<td style="width:119px;">22-362566</td>
			<td style="width:167px;">Any supplier can be used. Smaller tubes may be more effective for collection of small semen samples, such as from very small passerine birds like chipping sparrow.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge tubes</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1x PBS</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td>Used for storing semen samples prior to DNA extraction.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DL-Dithiothreitol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris base</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">10708976001</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EDTA</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">1233508</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">793566</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SDS</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">1614363</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAamp DNA micro kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">56304</td>
			<td>Qiagen QIAamp DNA micro kit is recommended specifically. This DNA extraction protocol has been attempted with other commercial kits but with little success. The reasons for this are unknown to us.&#160;</td>
		</tr>
	</tbody>
</table>

avian semen collection by cloacal massage and isolation of dna from sperm

정액의 컬렉션은 다양 한 정자의 품질, 정자 telomere 역학, 그리고 epigenetics 관련 된 연구를 포함 하 여 응용 프로그램에 대 한 유용할 수 있습니다. 새 생물학 연구에 널리 사용 되는 과목 이며 반복된 정자 샘플을 포함 하는 연구에 이상적입니다. 그러나, 몇 가지 리소스는 그 수집 및 조류 정액에서 DNA를 추출 하는 방법을 배울 하고자 현재 사용할 수 있습니다. 여기는 cloacal 마사지, 조류 정자를 수집 하기 위한 부드러운, 비-침략 적 수동 기술에 설명 합니다. 비록이 기술은 문학에서 설립, 그것은 사용 가능한 설명에서 배울 어려울 수 있습니다. 우리는 또한 상업적인 추출 키트를 사용 하 여 수정 하는 조류 정액에서 DNA를 추출에 대 한 정보를 제공 합니다. Cloacal 마사지는 생식 상태에서 어떤 중간 소형 남성 조류에 쉽게 사용할 수 있습니다. 컬렉션, 다음 정액 운동 성 분석에 대 한 즉시 사용 하거나 여기에 설명 된 프로토콜에 따라 DNA 추출에 대 한 동결 될 수 있습니다. 이 추출 프로토콜 조류 정자에 대 한 세련 되었고 여러 passerine 종 (통행 인 domesticus, Spizella passerina, Haemorhous mexicanus및 Turdus migratorius에서에서 수집 된 샘플에서 성공적으로 사용 되었습니다. )와 1 개의 columbid (비둘기 리비아).

Cloacal 마사지 그리고 DNA 추출을 사용 하 여 설명된 프로토콜 여러 passerine 종과 한 columbid (표 1)에서 수행 되었습니다. 정액 샘플 cloacal 마사지에 의해 수집 된에 정자의 존재는 400 배 확대 (그림 3)에서 복합 현미경 희석된 샘플의 작은 금액을 확인 하 여 확인 되었다. 집 참새는 여기 설명 된 프로토콜을 사용 하 여에서 수집 하는 정액 샘플...

Watch this Scientific Journal Video about Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm at JoVE.com

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

이 프로토콜 cloacal 마사지를, 조류, 그리고 수정 상업 추출 키트를 사용 하 여 새 정자에서 DNA 추출에서 정액을 수집 하는 비-침략 적 수동 기술에 설명 합니다.

조류 정액 컬렉션 Cloacal 마사지와 정액에서 DNA의 분리

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

avian-semen-collection-cloacal-massage-isolation-dna-from

Research

JoVE Journal

Biology

15.8K 조회수.  North Dakota State University. 이 프로토콜 cloacal 마사지를, 조류, 그리고 수정 상업 추출 키트를 사용 하 여 새 정자에서 DNA 추출에서 정액을 수집 하는 비-침략 적 수동 기술에 설명 합니다.

비디오: Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. 

Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female&#x2019;s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.

Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work.

수확 정자와 마우스의 인공 수정

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

연구

생물학

Isolation of Genomic DNA from Mouse Tails

마우스 뒷면에서 게놈 DNA의 분리

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

인간 췌장 아일릿 격리 : 부품 I : 소화 및 췌장 조직의 수집

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

조류 배아 판막병 전구 세포의 분리 및 문화

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

분리 및 체외에서</em의> 활성화 Caenorhabditis 엘레간스 정자

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

플라즈마 단백질 보존을위한 혈액 수집 장비로부터 게놈 DNA를 추출하는 실제적이고 새로운 방법

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

당연히 환경에서 Magnetotactic 박테리아를 발생의 수집, 절연 및 강화

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

마우스 모델에서 자궁 - 난관 배아 전송 및 정관

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

배양<em&gt; 예쁜 꼬마 선충</em &gt; 미세 폭격에 의한 무균 액체 미디어 및 형질 전환 웜의 생성에

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA 일렉트로, 근섬유의 분리 및 이미징

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

조류 정액 컬렉션 Cloacal 마사지와 정액에서 DNA의 분리

PLAYLIST