小鼠卵母细胞的细胞核移植到

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Summary

这部电影的协议,旨在帮助学习核转移。

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Egli, D., Eggan, K. Nuclear Transfer into Mouse Oocytes. J. Vis. Exp. (1), e116, doi:10.3791/116 (2006).

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Abstract

核移植到未受精的卵母细胞可以分化的细胞恢复发展潜力。这表明,底层的发育,分化和老化的过程,而不是遗传过程的后生。这些过程的可逆性打开令人兴奋的观点,在基础研究和在更遥远的未来,在再生医学。在小鼠胚胎干细胞可以从克隆胚胎植入前胚胎。这种胚胎干细胞会引起成人的有机体的所有细胞类型的能力。更重要的是,这些细胞是基因完全相同的捐助。如果适用于人类,这将允许从病人的干细胞的推导。然后将这些细胞可以分化成病人受影响的细胞类型,并研究在体外,或用来替换损坏或丢失的细胞。在小鼠核移植的研究仍然是重要的,因为它可以告知我们有关核重编程的原则。这部电影和所附议定书的目的是帮助学习在小鼠核移植,这种方法最初教授柳町(和歌山等,1998)集团开发的。

Protocol

准备工作:

  1. 一个超数排卵和胚胎培养microdrop的详细描述,可以发现其他地方的2。
  2. 准备好与胚胎培养基(如KSOM,Chemicon公司)microdrops培养皿。盖与矿物油(矿物油批次应与胚胎培养的兼容性测试)。平衡在37℃空气中加5%的CO 2中的C。 (热电子3110水套式孵化器或同等)
  3. 卵母细胞的隔离准备一道菜:HEPES广场滴缓冲CZB和透明质酸酶之一,在另一半的菜和HCZB的一半(0.1%W / V,Sigma公司)。封面与矿物油。保持菜上了一个解剖镜下加热阶段的温暖。
  4. 准备显微镜:放置一个μ约50米(略低于卵母细胞的直径更小)外径拿着吸管, μ一个吸管持有约20米的开放。控股移液器很容易做出必要的设备,针拉马(萨特仪器)和microforge(Narishige),但他们也可以从Humagen购买。

    3-5μ升汞装入一个微管(内径米)的尾巴。避免水银泄漏。准备与几个小滴11%W / V HCZB PVP(聚乙烯吡咯烷酮)和5μμg/ ml的细胞松弛素B在HCZB一盘菜。使用培养皿的盖子,而不是菜本身,盖子有一个很短的RIM不会干涉与控股和眼球摘除移液器运动。将拿着吸管HCZB下降和眼球摘除吸管。吸成控股吸管尖的小HZCB。
  5. 供体细胞的制备,在很大程度上取决于细胞类型,研究者打算执行的实验。在一般情况下,供体细胞应被视为与蛋白水解酶如胰蛋白酶,在冰上不停地减少,直到转移的粘性。
  6. 人道安乐死小鼠注射hCG后14-15小时。收获输卵管和剖析他们在温暖的HCZB透明质酸滴。经过约。 5分钟,卵母细胞大多是从卵丘细胞。然后,他们应该是在HCZB洗几次,然后进一步在预平衡的KSOM滴洗涤,并在孵化器中保存,直到眼球摘除。

眼球摘除

液压micromanipulators,如尼康TE200倒置显微镜(Narishige),处理完成。压电显微(Primetech)是用于钻透明带。平头眼球摘除和转移移液器,就可以买到从Humagen。清洁和润滑眼球摘除吸管滴在PVP的驱逐微观汞滴和吸气和驱逐的PVP。为了防止针的粘性,同样的程序应该做的各组,摘除或转让的卵母细胞之间。

小组的卵母细胞放入HCZB细胞松弛素B下降。本集团的大小应适应水平调查的经验。卵母细胞不应该保存在舞台上的时间超过20-30分钟。移动仪器HCZB细胞松弛素B滴。控股吸管和眼球摘除吸管应纳入与卵母细胞的赤道平面对齐。这将是可能的,如果拿着吸管外径略高于卵母细胞本身为小。直到可以看出不同的屈光中期主轴旋转一个卵母细胞。如果是初学者也可以看不到主轴,中期板可以识别DNA染料Hoechst公司和紫外光照射,但是,这是不兼容高效的胚胎发育。中期主轴在3点钟的位置,并保持控股吸管。应用压电脉冲穿透透明带。触摸眼球摘除吸管中期板。一旦中期主轴的电阻可以“感觉”,吸主轴和退出针。卵母细胞的细胞质是流动的,然而,中期主轴移动时,用移液管作为丛接触。尽量减少细胞质的量与主轴删除。经过集团已去核的卵母细胞,通过胚胎培养液中清洗,并放入孵化器中,直到他们转移。一些去​​核卵母细胞不应该被转移,但应该被激活,并作为一个眼球摘除控制。这些卵母细胞在几个小时内将片段,表明成功眼球摘除。

核移植

进入PVP溶液(11%PVP在HCZB)的细胞。如果在1 - 细胞阶段克隆逮捕,PVP浓度应降低至5%或更少。混合细胞以及机智h的PVP。拿起一个内径略大于细胞直径较小的吸管细胞。细胞膜破裂后的愿望。在某些情况下,重复驱逐和愿望,以及温柔的压电脉冲的应用可以被用来打破的供体细胞。一个新的供体细胞下降,应与各组卵母细胞被转移,成为一段时间后,供体细胞的粘性。断供体细胞注射后不久,打破了膜。为转移,集中在赤道平面上的卵母细胞本身,而不是赤道平面的透明带,并在同一平面上的位置转移和控股吸管。保持卵母细胞,包括其细胞质的一部分,坚决。使用压电脉冲穿透透明带。吸管尖把捐助细胞核,然后把枪头几乎到了对面的去核卵母细胞,拿着吸管,在卵母细胞深畦。移液器吸进极少量的卵母细胞的细胞质中,适用于一个单一的弱压电脉冲打破卵母细胞质膜,退出捐助核,迅速撤回针,而在胞质膜畦右端吸。同时撤回针吸,它应该有可能接近新台币留下的孔。这个“洞去除技术将减少,卵母细胞,溶解在NT的数目。在KSOM清洗重构卵母细胞和返回为1-3个小时的孵化器。

卵母细胞激活

准备一盘无钙MCZB飞沫和无钙MCZB加上10MM SR 2液滴的另一半的一半,5μμg/ ml的细胞松弛素B抑制极体挤压。平衡为30分钟。 5%以下,空气中一氧化碳2。在无钙MCZB和清洗新台币胚胎,然后放置在小群体,他们在不同滴钙免费MCZB。返回菜的孵化器和文化为5-6小时。为了控制人工激活协议和胚胎培养条件下,应使用非去核卵母细胞。他们将发展为孤雌生殖胚胎。激活之后,通过长期培养的胚胎在孵化器冲洗KSOM和地方胚胎。原核应在这个时间点可见,表明成功转让。

胚胎文化传媒食谱

主盐

开始在无菌1升瓶980毫升超纯H 2 O

干燥后放入组件:

盐4760毫克(81毫米)适马的S - 5886
氯化钾360毫克(5mm)的Sigma公司的P - 5405
硫酸镁 •7H 2 O 290毫克(1.18mM)Sigma公司的M - 2773
KH 2 PO 4 160毫克(1.17mM)Sigma公司的P - 5655
EDTA的2NA 40毫克(0.1毫米)适马E - 6635
葡萄糖(四)1000毫克(5.5毫米)适马的G - 6152

添加液体成分

NA -乳酸(乳酸)5.3毫升 西格玛44263
Pen'Strep 10毫升Gibco公司15140-122

对于MCZB库存盐:

过滤消毒500毫升主盐(适合3-4个月;储存于4 ° C)

对于HCZB库存盐:

开始用500毫升的主盐

添加50毫克,PVA(冷溶; SIGMA的P - 8136)

搅拌30-60分钟,无菌过滤器(3个月;储存于4 ° C)

+ +免费MCZB(卵母细胞激活在5%的CO 2)

开始用99毫升MCZB股票盐

干燥后放入组件:


碳酸氢钠3 211毫克适马的S - 5761

3毫克钠丙酮酸(丙酮酸)Sigma公司的P - 4562

L -谷氨酰胺15毫克适马的G - 8540

牛血清白蛋白(BSA)500毫克适马A - 3311

直到所有的组件涡流解散;无菌过滤器。

HCZB(大气环境显微)

开始用99毫升H​​CZB股票盐

干燥后放入组件:


HEPES -钠520毫克Sigma公司的H - 3784

碳酸氢钠3 42毫克适马的S - 5761

添加液体成分:

128毫米氯化钙2(18.8克/ L)1毫升Sigma公司的C - 7902

1 N盐酸调节pH值至7.5

直到溶解涡流;无菌过滤器。

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Discussion

祝你好运!

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Acknowledgments

DE想感谢他NT技巧和斯蒂芬沙利文博士和加勒特伯克霍夫共享协议的关键读博士隐藏阿久津。

Materials

Name Company Catalog Number Comments
Cytochalastin B Sigma-Aldrich 100x stock = 500 µg/ml Cytochalasin B in DMSO. Store at -20 ˚C.
Strontium Chloride 10x stock = 100 mM SrCl2 in H2O. Store at room temperature.
MCZB Stock Salts Filter sterilize 500 ml master salts (good for 3-4 months; store at 4 ˚C)
HCZB Stock Salts Start with 500 ml master salts. Add 50 mg PVA. Stir for 30-60 min and sterile filter (good for 3 months; store at 4 ˚C).
PVA Sigma-Aldrich P-8136 cold-soluble
HCZB with 11% w/v PVP Start with 20 ml HCZB medium in a 50 ml conical tube. Place 22 g PVP on top of liquid. Close tube and store undisturbed at 4 ˚C for 72 – 96 hr, at which time the PVP will have entered solution. Filter sterilize through an 8 mm filter and store at 4 ˚C.
PVP ICN Biomedicals MW 360,000
Please check for additional buffers compositions in the Protocol part.

DOWNLOAD MATERIALS LIST

References

  1. Eggan, K., Jaenisch, R. Mammalian and Avian Transgenesis- New Approaches. Pease, S., Lois, C. Springer. (2006).
  2. Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. Manipulating the mouse embryo. Press, C. S. H. L. (2003).
  3. Wakayama, T., Perry, A. C., Zuccotti, M., Johnson, K. R., Yanagimachi, R. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature. 394, 369-374 (1998).

Comments

6 Comments

  1. I am going to start ICSI in Equine (horese) oocyte and for this from start point I have some problems, in papers which I am going to follow they published that before injection they put 1 microlitter of sperm suspension in 3 microlitter of Sp-TALP containing 10% PVP (SIGMA) . I did the same, but you know 10% PVP (I calculated w/v) is to much powder that's why I found all of the sperm dead before injection. I don't know realy how I have to use this PVP (MW 360000) ?!

    Reply
    Posted by: Anonymous
    May 5, 2008 - 8:07 AM
  2. Hello - I work at Sigma-Aldrich in St. Louis, Mo. and have received an answer for your question from our technical service group.  It follows below: _____________________________________________________________________________ We really do not have an answer as we do not have an IVF product. We do have the following references however we have not researched them. Some references on this product are as follows: 1. Yamazaki, Y., et al., Reprogramming of primordial germ cells begins before migration into the genital ridge, making these cells inadequate donors for reproductive cloning. Proc. Natl. Acad. Sci. USA., 100(²1),, 1²²07-1²²1² (²003). ². Shitara, H., et al., Selective and Continuous Elimination of Mitochondria Microinjected Into Mouse Eggs From Spermatids, but Not From Liver Cells, Occurs Throughout Embryogenesis. Genetics, 156, 1²77-1²84 (²000). 3. Coy, J.F., et al., A complex pattern of evolutionary conservation and alternative polyadenylation within the long 3'-untranslated region of the methyl-CpG-binding protein ² gene (MeCP²) suggests a regulatory role in gene expression. Human Mol. Genet., 8(7), 1²53-1²6² (1999). It is commonly used in Denharts solution at 1% (10mg/ml). For mouse embryo culture, PVP 40 (40,000 mw) is used at 0.5% in the culture media. The PVP360,000 solution is used to immobilize or slow the sperm. You may want to use a 1% solution rather than a 10% solution. _____________________________________________________________________________ We hope that the above is helpful to you. - Diane

    Reply
    Posted by: Anonymous
    May 13, 2008 - 4:08 PM
  3. Do clones develop when injected into the body of the mother? ie not in the uterus? perhaps after a sufficient number of cell divisions, growth factors... has anyone tried? a little like conjointed twins. no be immune rejection if it dŒs, this could perhaps then allow to grow new members only, as happens often with conjointed twins. the new members would be fully young.

    Reply
    Posted by: Anonymous
    July 29, 2008 - 6:00 PM
  4. terrific, and very instructive. Thanks a lot. Yunhai from China.

    Reply
    Posted by: Anonymous
    December 25, 2008 - 3:40 AM


  5. Thanks a lot.

    YongWei from China.

    Reply
    Posted by: yongwei N.
    April 17, 2013 - 5:10 AM
  6. Hello,
    I'm a PhD student form Universitat Autònoma de Barcelona. The main tool of my studies is SCNT. I would like to contact with Dieter Egli and Kevin Eggan if it's possible, because i need some advices about the transfer of electrical impulse through the micropippete.
    Few months ago i used to use PVP 3% for cummulus cells, but i realised that PVP 10% was better for incresase the number of 2 cell embryos. Nowadays the problem is the transmition of piezzo. How much increases the PVP concentration, more difficult is the transmition of the electrical impulse across the micropippete. I've tried to increase the frequency, amplitude and duration of the electrical PiezoDrill, but at the moment is still too much difficult to breack the Zona pellucida.
    I'm sure that you will have some advices or an answer for this problem.
    Thank you.

    Reply
    Posted by: Laia P.
    December 22, 2014 - 1:27 PM

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