Ve Jeffrey Kittilson laboratuvarı için destek biz Jose Noguera cloacal Masaj, erken hakkında yardım almak için teşekkür ederiz. Bu eser bir ND EPSCoR FAR0022429 Ödülü BJH için tarafından desteklenmiştir. Yayın ücreti cömertçe NDSU bölümü Biyolojik Bilimler tarafından desteklendi.

Bu iletişim kuralı omurgalı hayvan konular içerir ve kurumsal hayvan bakım ve kullanım Komitesi (IACUC) Kuzey Dakota Eyalet Üniversitesi tarafından onaylandı.
1. meni koleksiyonundan bir ötücü kuş cloacal masaj kullanarak
Not: Cloacal masaj bir çoğalma etkin kuşlar sadece üzerinde etkili meni koleksiyonu tekniktir, ancak başarıyla uygun esir durumlarda Üreme mevsimi dışında yapılan. Üreme faaliyet cloacal masaj kullanmadan önce hedef türler için tespit edilmelidir. Bu teknik yakalama üzerine yabani kuş vurmak ya da esaret altında düzenlenen kuşlar kullanılabilir.
<ol><li>(İçinde durumu doğurmak) bir erkek kuş ile başlayan sigara dominant el pençesinde bander'ın aktarım kuş dominant el palm (şekil 1) dokunmadan ventral yan ile.<ol>
<li>Kuş başı lateral palmiye kenarına en yakın olduğundan emin olun. Hafif baş ve orta parmak, yüzük ve serçe kullanarak güvenli. Bu kuşun havalandırma ve kuyruk maruz bırakır.</li>
		<li>Kuş bacaklar gevşek veya hafifçe ölçülü palmiye kullanarak bırakın.</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55324/55324fig1.jpg"> Resim 1 : Kavrama cloacal masaj için. Kuş hafifçe dominant el palm ve pinky parmak tarafından güvenli baş dokunmadan ventral yan ile sabitlenir. <a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<ol start="2"><li>Baskın işaret parmağı ve başparmak lağım üstün sonu her iki tarafında yerleştirin ve hafifçe parmak birlikte kuyruk ( Şekil 2) doğru çok hafif basınç uygularken çimdik. Lağım cloacal mukoza (pembe iç) açığa çıkar. Not: kuş ise cloacal tümsek küçük olabilir şu anda meni, üreten ve lağım evert zordur. Tecrübesiyle, çoğalma etkin ve etkin olmayan erkek5,11cloacas arasında ayırt etmek kolaydır.</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55324/55324fig2.jpg"> Resim 2 : Parmak pozisyonu ve cloacal tümsek. Lağım üstün sonuna (A) hafif basınç uygulayarak çıkar. Bir çoğalma aktif erkek ötücü içinde cloacal tümsek açıktır ve firma (B). <a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig2large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<ol start="3"><li>Sigara dominant el ile kuyruk el stabilize etmek için tabanının dorsal tarafta parmağı yastık yerleştirin ve diplidir lağım hemen altında parmak ucu yerleştirin. Not: Kısa ve yumuşak küçük resim cloacal masaj gerçekleştirmek için avantajlıdır ve kaza sonucu yaralanma kuş korur.</li>
	<li>Baskın başparmak hareket derin ve cranially lağım kalma orta basınç ile tekrarlayan bir hareket. Kuş cinsel ilißki kadar devam edin. Bireysel bağlı olarak, bu genellikle 1 ve birkaç düzine vuruşları arasında sürer.</li>
	<li>En kısa zamanda kuş cinsel ilißki, meni mikrohematokrit tüp içinde toplamak. Cloacal masaj birden fazla cinsel ilißki elde etmek için yineleyin. Not: Ötücü meni hafif kahverengi ve genellikle biraz kalın bir tutarlılık vardır. Beyaz ve koyu maddelerden lağım atılır meni değildir ve meni örnek kontamine. Az miktarda kan lağım bulunan görünümünü veya içinde ile karışık meni genellikle masaj sırasında çok fazla baskı tarafından neden olur.</li>
	<li>DNA ekstraksiyon için 1 x PBS (pH 7,4) 20 µL ile microcentrifuge tüp meni yerleştirin. Not: 1 x PBS içinde depolanan meni suni tohumlama için uygun değildir, ancak DNA ekstraksiyon daha sonraki bir tarihte kullanılan-80 ° C'de saklandığında PBS Oda sıcaklık olabilir veya soğutulmuş ama meni örnek yatırılır sıvı bir durumda olması gerekir.<ol>
<li>Sperm örnek varlığı seyreltilmiş örnek (şekil 3) bileşik mikroskop altında küçük bir miktar görüntüleyerek doğrulayın.</li>
	</ol>
</li>
	<li>-80 ° C'de seyreltilmiş meni ayıklama kadar saklayın.</li>
</ol>2. çıkarma DNA'ın kuş meni
Not: Bu iletişim kuralı birden fazla ticari DNA ekstraksiyon kitleri ile test edilmiştir, ancak sadece bir DNA kiti12ile kullanıldığında başarılı oldu. Değiştirilen kiti Protokolü adım simgesiyle *.
<ol><li>Dithiothreitol (DTT) suda 1 M çözeltisi hazırlamak ve bir vortex.* kullanarak mix<ol>
<li>Aliquot DTT çözüm ve -20 ° c kadar kullanımdan hemen önce saklayın. Elde edilebilir için her örnek, 1 M DTT solution.* 10 µL gerektirir</li>
	</ol>
</li>
	<li>20 mM Tris-Cl, pH 8.0 içeren bir arabellek hazırlamak; 20 mM EDTA; 200 mM NaCL; ve % 4 SDS. Bu buffer.* 90 µL ayıklanmasını her örneği gerektirir<ol>
<li>Arabellek, oda sıcaklığında saklayın. Çöktürülmüş Eğer o dissolve.* 56 ° c sıcak</li>
	</ol>
</li>
	<li>Bir rocker veya shaker 65 içeren bir kuluçka Onceden ° C.*</li>
	<li>Hemen çıkarma işlemine başlamadan önce 10 µL DTT çözüm 10 örnekleri açılan Eğer adım 2.2 her örnek elde edilebilir, örneğin 900 µL arabellek artı 100 µL DTT çözüm için hazırlanan tampon 90 µL ile karıştırın. Bu karışım DTT-buffer.* adı verilecek</li>
	<li>70 µL 1 x PBS tüm seyreltilmiş meni örnek ve iyi bir vortex.* kullanarak karıştırın</li>
	<li>100 µL eklemek DTT-tampon ve 10 µL İndinavir K çözüm örnek. Girdap 20 saniye sonra bir rocker veya shaker.* 1 saat önceden ısıtılmış kuluçka makinesine kuluçkaya</li>
	<li>200 µL tampon AL ve 200 µL eklemek taze alkol (% 100) ve 20 seconds.* için girdap</li>
	<li>Tüm örnek bir spin sütuna aktarmak ve 6.000 x g. atma 1 dakikada toplama tüp santrifüj kapasitesi.</li>
	<li>Sütun yeni bir tüp yerleştirin ve 500 µL hazırlanan tampon AW1 ekleyin. 1 6.000 x g. atma dakikada toplama tüp santrifüj kapasitesi.</li>
	<li>Sütun yeni bir tüp yerleştirin ve 500 µL hazırlanan tampon AW2 ekleyin. 6.000 x g. adlı 1 dakika santrifüj atmak akışı aracılığıyla ve sütun geri televizyonda yer.</li>
	<li>3 dakika 20.000 x g. atma, koleksiyon tüp santrifüj kapasitesi ve sütun bir 1.5 mL microcentrifuge tüp yerleştirin.</li>
	<li>Damlalıklı 35 AE µL arabellek sütun membran doğrudan ve oda sıcaklığında 5 güncellemeler için kuluçkaya</li>
	<li>1 dakikada 14.000 x g. atma spin sütun santrifüj kapasitesi ve ayıklanan örnekleri-80 ° C'de depolayın</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>

Yazarlar onlar rakip hiçbir mali çıkarları var bildirin.

Biz küçük ve orta ölçekli kuşlar üreme etkin durumda sperm toplama ve kuş sperm DNA ayıklanması için basit ve güvenilir bir yöntem açıklanmaktadır.
Açıklanan Sperm DNA ekstraksiyon protokolü bir kit basitlik için değiştirilebilir, ancak kuş meni kullanmak için rafine. Sperm lizis standart ayıklama kimyasallar9,13tarafından dayanıklı ve sperm örneği ötücüler toplanan oldukça küçük (genellikle bir ka?...

Sperm kalitesini ve rekabet1, sperm telomer dynamics2, epigenetik3ve benzer konular, ilgili sıra onlar biyolojik araştırmalarda yaygın olarak kullanılır ve sperm kolayca cloacal kullanarak tatmak mümkündür çalışmaları için ideal konular kuştur Masaj. Cloacal masaj meni kuşlar4,5,6toplamak için bir nazik, non-invaziv manuel tekniktir. Yinelenen örnekleri kolayca elde edilebilir ve hiçbir özel araç alan veya laboratuvar gerçekleştirmek basit yapmak gerekli. Cloacal masaj yıllardır kullanılıyor olsa da, mevcut gelen açıklamalar yazılı öğrenmek zordur. Bu yayın belirsizlik cloacal masaj öğrenmede yer ve zamanı azaltmak için tasarlanmıştır.
Meni cloacal masaj kullanarak kuşlardan toplanan hemen hareketliliği değerlendirme4,7 veya suni tohumlama8için kullanılan veya gelişmiş görüntüleme ve DNA ekstraksiyon gibi diğer kullanımlar için dondurulmuş. Sperm örneği ötücü kuşlar küçük ama yoğun paketlenmiş sperm barındırır. DNA sperm9özel koruyucu özellikleri üstesinden gelmek için değişiklikleri ile sadelik için bir ticari ekstraksiyon kiti kullanılarak elde edilir. Çıkarma sonra sperm telomerlerin qPCR10kullanılarak ölçülebilir.

<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

Meni topluluğu çeşitli sperm kalitesi, sperm telomer dynamics ve epigenetik ilgili çalışmalar da dahil olmak üzere uygulamalar için yararlı olabilir. Kuşlar biyolojik araştırmalarda yaygın olarak kullanılan konulardır ve tekrarlanan sperm örnekleri içeren çalışmaları için idealdir. Ancak, az kaynak şu anda toplamak ve kuş sperm DNA ayıklamak nasıl öğrenmek isteyenler için kullanılabilir. Burada cloacal Masaj, kuş sperm toplamak için bir nazik, non-invaziv el ile teknik açıklayın. Ne kadar bu teknik literatürde kurulur, kullanılabilir açıklamaları öğrenmek zor olabilir. Ayrıca DNA kuş meni bir ticari ekstraksiyon kiti değişiklikleri ile kullanarak çıkarma hakkında bilgi sağlar. Cloacal masaj kolayca herhangi bir küçük ve orta boyutlu erkek kuş üreme durumda kullanılabilir. Koleksiyon meni hemen motilite testleri için kullanılan, veya burada açıklanan protokol sonrası DNA ekstraksiyon için dondurulmuş. Bu ayıklama protokolü için kuş sperm rafine ve başarıyla birçok ötücü türü (Passer domesticus, Spizella passerina, Haemorhous mexicanusve Turdus migratorius toplanan örnekleri üzerinde kullanılan ) ve bir columbid (Columba livia).

Cloacal masaj ve açıklanan protokol birçok ötücü türü ve bir columbid (Tablo 1) gerçekleştirilen ayıklama DNA'yı kullanarak. Sperm meni örneklerinin cloacal masaj tarafından toplanan varlığı 400 X büyütme (şekil 3) seyreltilmiş numune bir bileşik mikroskop altında küçük bir miktar ile ilgilenen tarafından doğrulandı. DNA sperm örneği açılan house burada açıklanan protokolü kullanarak serçeler toplanan sonr...

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

Bu iletişim kuralı cloacal Masaj, kuşlar ve kuş sperm bir ticari ekstraksiyon kiti değişiklikleri ile kullanarak çıkarma DNA meni toplamak için bir non-invaziv el ile teknik açıklar.

Kuş sperm toplama Cloacal masaj ve Sperm DNA izolasyonu

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 Görüntüleme.  North Dakota State University. Bu iletişim kuralı cloacal Masaj, kuşlar ve kuş sperm bir ticari ekstraksiyon kiti değişiklikleri ile kullanarak çıkarma DNA meni toplamak için bir non-invaziv el ile teknik açıklar.

Video: 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.

Farelerin Hasat Sperm ve Suni Tohumlama

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 ...

Biyoloji

Isolation of Genomic DNA from Mouse Tails

Fare Tails Genomik DNA izolasyonu

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.

İnsan Pankreas Adacık İzolasyonu: Bölüm I: Sindirim ve Pankreas Doku Koleksiyon

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.

Kuş Embriyonik Kapak Progenitör Hücreler izolasyonu ve Kültür

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.

İzolasyon ve In vitro Etkinleştirme 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 ...

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.

Plazma Protein Koruma Kan Alma Kitleri Genomik DNA Ayıklama Pratik ve Yeni Bir Yöntem

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.

Doğal Çevre gelen Magnetotactic Bakteri meydana gelen toplanması, İzolasyon ve Zenginleştirme

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.

Fare Modelinde utero-tubal Embriyo Transferi ve Vazektomi

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.

Kültürleme<em&gt; Caenorhabditis elegans</em &gt; Mikroparçacık bombardımanı ile aksenik Sıvı Medya ve Transgenik Worms Yaratılış

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 Elektroporasyon, Myofibers İzolasyon ve Görüntüleme

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

Kuş sperm toplama Cloacal masaj ve Sperm DNA izolasyonu

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