여기에 제시 연구는 찰스 강에 의해 지원되었다. 저자는 동물 보호와 호르몬 주사를위한 유전자 조작 모델 및 서비스 그룹과 발생학 직원에게 가장 감사하고 있습니다.

1. 마우스 정자 cryopreservation <ol><li> cryopreserved하는 각 라인에 대한, 시작하기 전에 다음 사항을 준비합니다. <ol><li> 키트 CPA의 2백40리터과 4 잘 요리 잘 하나 </li><li> 다음과 같이 15 정자 cryopreservation의 빨대 <ol><li> 주사기에 가장 가까운 면화 플러그 하나 ML 주사기로 0.25 ML 빨대를 연결합니다. 각 짚으로 다음 IVF 매체의 8cm (약 160 μl), 공기의 1cm를로드합니다. 보존하는 라인의 이름으로 빨대를 분류. </li></ol></li></ol></li><li> 아까 2 폼 상자에 깊이 15cm해야합니다. 뚜껑을 닫습니다. </li><li> cryopreserved 수 각 라인에서 2 수컷 쥐를 안락사. 마우스는 10 및 60 주 된 사이 여야합니다. 각 마우스의 중이야 deferens과 함께 두 개의 꼬리 epididymides를 제​​거합니다. </li><li> 이전에 준비한 4 잘 요리 (단계 1.1)의 잘 CPA 함유로 epididymides를 놓습니다. </li><li> 5-7 세로 각 epididymis의 상처와 부드럽게 만들기정자를 밀어 각 중이야의 deferens를 당겨요. </li><li> 3~5분에 대한 실온에서 부드럽게 손으로 CPA와 epididymis 혼합물을 흔들어. 이것은 정자가 수영을하실 수 있습니다. </li><li> 이전에 준비 빨대를 사용하여 정자 서스펜션의 5mm를 (약 10 μl) 대기음 그리고 공기의 1cm. </li><li> 15 빨대 총까지이 과정을 반복합니다. 로드와 최고의 정자 생존을위한 10 분 이내에 빨대를 밀봉. </li><li> 조심스럽게 빨대의 양쪽을 밀봉 가열. 먼저 정자 칼럼과 냉동 용기에 빨대를 놓습니다. </li><li> 손잡이를 연장 용기에 키트의 일부로 제공 제 1 ML의 피펫을 첨부합니다. 뚜껑에있는 구멍을 통해 거품 상자에 아까 2 용기를 놓으십시오. 10 분 후면 2 세로 부동에게 용기를 허용합니다. </li><li> LN2에 용기를 담가. 정자는 현재 cryopreserved되고 장기 에선이 스토리지로 이동하실 수 있습니다. </li><li> 용기는 사용하지 않아야다음 라인은 그 때까지 실내 온도 예열합니다. </li></ol> 2. 마우스 superovulation <ol><li> 일반적으로 정자 기증자와 같은 배경의 젊은 여성 생쥐를 사용합니다. </li><li> 마우스는 ~ 12g (세 3-4주)하여야한다. </li><li> 임신 암말 혈청 gonadotropin (PMSG)의 50-10 IU와 intraperitoneal 주사를 수행합니다. </li><li> 46-48시간 기다립니다. </li><li> 인간 chorionic gonadotropin (hCG)의 50-10 IU와 intraperitoneal 주사를 수행합니다. </li><li> 아래에 설명된대로 hCG 주사 후 수확 oocytes가 14-16 H. </li></ol> 3. 마우스 정자 복구 및 IVF <ol><li> IVF 전에 해동하는 각 빨대에 대해, 37 ° C CO 2 배양기에서 적어도 한 시간 동안 다음을 준비하고 평형. <ol><li> 정자의 배양 접시 하나 35mm 페트리 접시, 기름으로 덮여 정자 치료 매체 (STM)의 90 μl 드롭과 함께. </li><li> 접시를 절단 : oocyte 컬렉션 10 s의 최대의uperovulated 여성, IVF 매체 3 ML 하나의 35mm 페트리 접시. </li><li> IVF 잘 : IVF 5 superovulated 여성, IVF 미디어 500 μl (... 당신은 10 superovulated 암컷을 사용하는 경우, 예를 들어, 2 우물이 필요합니다)와 함께 4 잘 요리 잘 한 각 그룹에 대한 </li><li> 사용하는 4 잘 IVF 요리의 각 잘 세척을위한 칼륨 단순 3 ML 최적화된 미디어 (KSOM) 하나의 35mm 페트리 접시 : 접시를 세척 </li><li> 문화 요리 : IVF 요리의 각 우물에서 배아 문화의 기름으로 덮여 KSOM 매체 50 μl 방울 한 35mm 페트리 접시. </li></ol></li><li> 후면 2 밀짚 후 2-3 분 동안 37 ° C의 물을 욕조에서 장소를 제거합니다. </li><li> 물 목욕에서 짚을 제거하고 여분의 물을 제거하는 닦아냅니다. 면화 플러그 가까운 최초의 공기 열에 가까운 첫 번째 절개를합니다. 여전히 정자 칼럼 전에 보이는 공기 칼럼있을 것입니다 IVF 미디어에서 짚을 잘라. </li><li> 다음의에서 빨대의 끝을 잘라econd 공기 열, 정자 열을 절단되지 않도록주의하고. 45 각도로 절단 - 이것은 정자를 대기음하는 것이 훨씬 쉽게됩니다. </li><li> 200 μl pipettor와 빨대의 말초 끝에서 정자 현탁액을 기음. </li><li> STM 드롭의 중심에있는 정자 현탁액을 놓고 37 ° C 5 % CO 2 배양기에서 45~60분을위한 요리를 품어. </li><li> 정자 보육 동안 절단 접시에 superovulated 여성의 oviducts을 해부하다. 적운 - oocyte의 단지 (COCs)를 발표, 각 수란관를 조각조각 30g 바늘이나 집게를 사용합니다. 더러운 미디어가 추가 세척 단계를 만들면서 IVF 미디어의 지방과 혈액을 피하십시오. </li><li> 일단 모든 oviducts가 찢어진되었습니다, 5 암컷에서 각 IVF 우물에 COCs을 전송. superovulation에 의해 만들어진 COCs의 수는 매우 의존 변형입니다. </li><li> STM 드롭의 주변에서 정자의 10-15 μl를 수집 한 IVF 거름을 잘 약 14-16시간은 hCG inj을 게시ection. 각 잘 사용에 대한 수정을 반복합니다. 최종 정자 농도는 약 1,000,000 / ML 것입니다. </li><li> 37 ° C 5% CO 2 배양기에서 4~6시간에 대한 Coculture의 정자와 oocytes. </li><li> 37 ° C 5% CO 2 배양기에서 개발 KSOM의 방울에 2 회 문화 KSOM 최소 배아를 씻으십시오. </li><li> IVF 속도는 밖으로 야간 문화 이후에 사용되는 총 oocytes의 두 세포 단계의 배아로 계산되었다. </li></ol> 4. 대표 결과 5 찰스 리버 마우스 근친 계통에서 정자가 냉동했습니다. 보호 비율은 신선한 정자 평가 가치에 의해 냉동 정자 평가 가치를 나누어 계산됩니다. 정자 운동성의 보호 비율은 각각 60.2 %, 81.3 %, 78.6 %, 66.5 %, C57BL/6NCrl, 129S2/SvPasCrl, FVB / NCrl, DBA/2NCrl 및 BALB / cAnNCrl에 대한 61.0 %, 있었다. 빠른 진보 운동성에 대한 보호 비율은 47.3 %, 69.4 %, 57.0 %, 52.8 %, C57BL/6NCrl, 129S2/SvPas에서 54.1 % 있었다<html> CRL, FVB / NCrl, DBA/2NCrl, 각각 BALB / cAnNCrl (그림 1). 냉동 - 해동 정자로 IVF 요금은 55.7 %, 26.4 %, 87.3 %, 87.8 % 각각 26.2 C57BL/6NCrl에서 % 129S2/SvPasCrl, FVB / NCrl, DBA/2NCrl 및 BALB / cAnNCrl, (그림 2)되었습니다 . 1.5 년 동안, 49 GM 마우스 라인 정자 cryopreservation에 의해 보관되었습니다. 이 라인은 나중에 성공적으로 네 여성의 종자에서 IVF (C57BL / 6, BALB / C, DBA / 1 129Sv) (그림 3)에서 (로 태어나 살고 새끼에 의해 정의된) 복구되었습니다. <img alt="그림 1." src="/files/ftp_upload/3713/3713fig1.jpg" /> 그림 1. 마우스의 정자 운동성 및 신속한 프로 그레시브 운동성의 보호 비율 <img alt="그림 2." src="/files/ftp_upload/3713/3713fig2.jpg" /> 마우스에서 냉동 - 해동 정자와 함께 그림 2.에서는 시험 관내 수정 3713fig3.jpg &quot;/&gt; 그림 3. GM의 마우스에서 냉동 - 해동 정자와 체외 수정을의

<ol>
	<li>Tada, N., Sato, M., Yamanoi, J., Mizorogi, T., Kasai, K., Ogawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+mouse+spermatozoa+in+the+presence+of+raffinose+and+glycerol.">Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol.</a> J. Reprod. Fertil. 89, 511-516 (1990).</li><li>Yokoyama, M., Akiba, H., Katsuki, M., Nomura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+normal+young+following+transfer+of+mouse+embryos+obtained+by+in+vitro+fertilization+using+cryopreserved+spermatozoa.">Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa.</a> Jikken Dobutsu. 39, 125-128 (1990).</li><li>Sztein, J. M., Farley, J. S., Young, A. F., Mobraaten, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+of+cryopreserved+mouse+spermatozoa+affected+by+temperature+of+collection+and+rate+of+thawing.">Motility of cryopreserved mouse spermatozoa affected by temperature of collection and rate of thawing.</a> Cryobiology. 35, 46-52 (1997).</li><li>Thornton, C. E., Brown, S. D., Glenister, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+numbers+of+mice+established+by+in+vitro+fertilization+with+cryopreserved+spermatozoa:+implications+and+applications+for+genetic+resource+banks,+mutagenesis+screens,+and+mouse+backcrosses.">Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens, and mouse backcrosses.</a> Mamm. Genome. 10, 987-992 (1999).</li><li>Sztein, J. M., Farley, J. S., Mobraaten, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+fertilization+with+cryopreserved+inbred+mouse+sperm.">In vitro fertilization with cryopreserved inbred mouse sperm.</a> Biol. Reprod. 63, 1774-1780 (2000).</li><li>Liu, L., Nutter, L. M., Law, N., McKerlie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+freezing+and+in+vitro+fertilization+in+three+substrains+of+C57BL/6+mice.">Sperm freezing and in vitro fertilization in three substrains of C57BL/6 mice.</a> J. Am. Assoc. Lab. Anim. Sci. 48, 39-43 (2009).</li><li>Nakagata, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+mouse+spermatozoa+and+in+vitro+fertilization.">Cryopreservation of mouse spermatozoa and in vitro fertilization.</a> Methods. Mol. Biol. 693, 57-73 (2011).</li><li>Mazur, P., Koshimoto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+intracellular+ice+formation+the+cause+of+death+of+mouse+sperm+frozen+at+high+cooling+rates.">Is intracellular ice formation the cause of death of mouse sperm frozen at high cooling rates.</a> Biol. Reprod. 66, 1485-1490 (2002).</li><li>Jin, B., Yamasaki, C., Yamada, N., Seki, S., Valdez, D. M., Kasai, M., Edashige, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+by+which+mouse+spermatozoa+are+injured+during+freezing.">The mechanism by which mouse spermatozoa are injured during freezing.</a> J. Reprod. Dev. 54, 265-269 (2008).</li><li>Visconti, P. E., Westbrook, V. A., Chertihin, O., Demarco, I., Sleight, S., Diekman, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+signaling+pathways+involved+in+sperm+acquisition+of+fertilizing+capacity.">Novel signaling pathways involved in sperm acquisition of fertilizing capacity.</a> J. Reprod. Immunol. 53, 133-150 (2002).</li><li>Ostermeier, G. C., Wiles, M. V., Farley, J. S., Taft, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserving,+distributing+and+managing+genetically+modified+mouse+lines+by+sperm+cryopreservation.">Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation.</a> PLoS ONE. 3, e2792-e2792 (2008).</li></ol>

저자는 찰스 리버,이 키트의 제조 업체의 직원입니다.

1990 년 이후 18 %의 raffinose 3 % 탈지 우유를 사용하여 기본적인 정자 동결 프로토콜은 마우스의 여러 변종에 신뢰성이 입증되었으며 마우스 라인의 다수는 1,2,3,4,5,6,7를 cryopreserved되었습니다. 동결 융해 과정 동안 정자 세포 손상의 주요 원인은 얼음 형성 8,9 및 반응 산소 종의 10과 관련된되었습니다. 냉동 매체 등 monothioglycerol 등의 아미노산과 같은 자유 라디칼 청소부, 그리고 감소 에이전트, 함께 보완은 조사와 크게 C57BL / 6 11,6 포함하여 근친 계통에서 냉동 - 해동 정자로 IVF 속도를 증가 입증되었습니다. 현재 프로토콜에서, 우리는 상용 산화 억제제와 얼음 차단제와 함께 기본 CPA를 최적화하여하고 정자 동결 IVF 절차를 수정하여 마우스 정자 cryopreservation을위한 새로운 I • Cryo 키트를 개발했습니다. 정자 운동성의 보호 비율(&gt; 60 %) 및 ​​신속한 프로 그레시브 운동성은 (&gt; 45 %) I • Cryo 키트 냉동 오 근친 마우스 종자의 정자에 대한 보였다. 5 마우스 근친 계통에서 냉동 - 해동 정자와 함께 2 - 세포 배아 단계 개발 속도는 현저하게 기본 CPA 11,6과 그에 비해 향상되었다. 키트를 사용하여, 사용자는 각 라인 2 남성의 냉동 정자로 정자 집중 서스펜션을 만듭니다. 단 냉동 15 빨대으로 과정이 빠르고, 쉽고, 단기 또는 장기 저장을위한 더 적은 저장 공간을 차지합니다. 대부분의 시간은, 마우스 라인은 1 개 또는 2 개 빨대를 해동하여 복구할 수 있습니다.

<table border="1">
	<tbody>
		<tr>
			<th>
				Materials</th>
			<th>
				Supplier</th>
			<th>
				Catalogue number</th>
			<th>
				Comments</th>
		</tr>
		<tr>
			<td>CPA</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>STM</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>IVF medium</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>0.25 ml plastic straw</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Sperm freezing canister</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Cane and goblet</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M5310</td>
			<td>Should be embryo tested</td>
		</tr>
		<tr>
			<td>KSOM medium</td>
			<td>Millipore</td>
			<td>MR-106-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm Petri dish</td>
			<td>Falcon</td>
			<td>351008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heat sealer</td>
			<td>ABTEC</td>
			<td>TISH-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4-well multidish</td>
			<td>Nunc</td>
			<td>73521-424</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>Gilson</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37&deg;C + 5% CO2 incubator</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LN2 storage system</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37&deg;C water bath</td>
			<td>VWR</td>
			<td>89032-196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>hCG</td>
			<td>Intervet</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>EMDChemicals</td>
			<td>367222</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1cc syringe w/ 26G 3/8 needle</td>
			<td>BD</td>
			<td>BD309625</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5602</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30 gauge &frac12; needle</td>
			<td>BD</td>
			<td>305106</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Roboz</td>
			<td>RS-6802</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz</td>
			<td>RS-5135, RS-5110, RS-5005</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

mouse sperm cryopreservation and recovery using the i&middot;cryo kit

마우스의 새로운 유전자 변형 (GM) 종자의 수천 transgenesis와 녹아웃 기술의 출현 이후 개발되었습니다. 이러한 귀중한 동물의 대부분은 긴급의 경우에도 백업 계획, 살아있는 동물로만 존재합니다. 배아의 Cryopreservation이 백업을 제공하지만 비용이 많이 드는 수 긴 절차 수 있으며, 일반적으로 성공을 위해 동물의 많은 요구하실 수 있습니다. 마우스 정자가 성공적으로 18%의 raffinose 3 % 탈지 우유로 구성된 기본 cryoprotective 에이전트 (CPA)와 cryopreserved 수있다는 발견 이후, 정자 cryopreservation는 보관 배포 이러한 귀중한 종자의 복구를 위해 허용하고 비용 효율적인 절차되고있다 . 여기 마우스 정자 cryopreservation을 위해 새로 개발된 I • Cryo 키트를 보여줍니다. 타고난 생쥐 다섯 일반적으로 사용되는 종자의 정자는이 키트를 사용하여 동결 후 회복되었다. 정자 운동성 높은 보호 비율 (&gt;60 %)과 제어 (기본 CPA)에 비해 빠른 진보적인 운동성은 (&gt; 45 %) 5 근친 마우스 변종이 키트와 함께 냉동 정자에 대한 보였다. 복구 정자로 IVF 후 두 세포 단계의 배아 개발은 검사 모든 5 마우스 종자에 지속적으로 개선되었다. 1.5 년 동안, 49 GM 마우스 라인은 I • Cryo 키트와 정자 cryopreservation에 의해 보관했다 나중에 IVF로 회복되었습니다.

Watch this Scientific Journal Video about Mouse Sperm Cryopreservation and Recovery using the IÃ‚Â·Cryo Kit at JoVE.com

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

여기 마우스 정자 cryopreservation을 위해 새로 개발된 I • Cryo 키트를 보여줍니다. 냉동 - 해동 정자와 두 개의 세포 단계의 배아 개발이 키트의 사용 5 마우스 종자에 지속적으로 개선되었다. 1.5 년 동안, 49 유전자 변형 마우스 라인 전 • Cryo 키트와 정자 cryopreservation에 의해 보관했다 나중에 성공적으로 IVF로 회복되었습니다.

전 · Cryo 키트를 사용하여 마우스 정자 Cryopreservation 및 복구

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

mouse-sperm-cryopreservation-and-recovery-using-the-icryo-kit

Research

JoVE Journal

Biology

39.5K 조회수.  Charles River. 여기 마우스 정자 cryopreservation을 위해 새로 개발된 I • Cryo 키트를 보여줍니다. 냉동 - 해동 정자와 두 개의 세포 단계의 배아 개발이 키트의 사용 5 마우스 종자에 지속적으로 개선되었다. 1.5 년 동안, 49 유전자 변형 마우스 라인 전 • Cryo 키트와 정자 cryopreservation에 의해 보관했다 나중에 성공적으로 IVF로 회복되었습니다.

비디오: Mouse Sperm Cryopreservation and Recovery using the I·Cryo Kit

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

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Qiagen Miniprep 키트를 사용하여 세균의 플라스미드 식민지에서 DNA를 정화

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Immunohistochemistry : 벡터 연구소에서 Vectastain ABC 키트를 사용하여 파라핀 섹션

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

바이오 - 래드 총 RNA 키트를 사용 Neuroprecursor 셀에서 RNA 추출

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

시그마의 압축을 풉니다 - N - 앰프 조직 PCR 키트를 사용하여 마우스 조직의 신속한 Genotyping

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

수집 및 햄스터 Oocytes 및 마우스 태아의 Cryopreservation

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

높은 처리량 Zebrafish의 정자 Cryopreservation에 대한 방법과 체외에서 수정

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

에틸렌 글리콜 기반 Vitrification에 의한 마우스 태아의 Cryopreservation

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

유도 만능의 줄기 세포에서 파생 마우스의 생성

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

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

생선 정자 평가 소프트웨어를 사용 하 여 및 냉각 장치

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...