6.5 DPC小鼠胚胎剖析

Biology

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Summary

植入后胚胎的隔离允许一个研究基因的图案和分析细胞系的胚胎发育过程中的决策过程,但早期胚胎的正常清​​扫,可以是具有挑战性的。这个协议描述为一个方法,隔离早期胚胎原始连胜阶段(〜6.5天coitum [DPC])。

Cite this Article

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Shea, K., Geijsen, N. Dissection of 6.5 dpc Mouse Embryos. J. Vis. Exp. (2), e160, doi:10.3791/160 (2007).

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Abstract

在哺乳动物胚胎发育的早期阶段中基因表达模式的分析可以提供有关基因的功能,细胞与细胞间的相互作用和信号传导机制,引导胚胎图案的重要线索。然而,从小鼠胚胎植入后不久蜕膜剥离可以是一个具有挑战性的的过程,缺乏详细的一步一步的这个过程中的文档。

在这里,我们将演示如何植入后(6.5 DPC)的胚胎首先剖析了孕鼠的子宫(阴道塞被指定每天0.5 poist coitum检测),随后解剖胚胎从母体蜕膜的隔离。赖克特的膜的夹层以及拆除ectoplacental锥描述。

Protocol

从雌性小鼠生殖器官的解剖

  1. 怀孕的动物安乐死的CO 2,使用压缩气体窒息。
  2. 莱吸水垫的动物仰卧浸泡在70%的乙醇,以减少污染解剖与鼠毛的风险。
  3. 捏皮肤,做一个小切口与常规手术剪刀中线外侧。牢牢把握的上方和下方的切口的皮肤,除了拉向头部和尾部的皮肤暴露腹部。
  4. 掌握与钳腹膜切开暴露腹腔。
  5. 在体腔的背地区找到生殖器官:两个子宫角,输卵管和卵巢。

原条阶段(6.5 DPC)夹层

  1. 删除抓下面的子宫输卵管子宫角和削减沿mesometrium。放置在冰冷的PBS的解剖子宫。

    图1

    图1

  2. 分开来每个胚胎切割之间沿子宫角植入网站。

    图2

    图2


  3. 抓住钟表镊子滑动与周围的肌肉层和笼罩蜕膜组织的肌肉子宫内膜。后面的肌肉层剥离,露出蜕膜。

    图3

    图3

  4. 在心尖(约1 / 5的蜕膜组织),这将暴露midventral或远端尖封闭胚胎暴露蜕膜部分剪掉。

    图4

    图4

  5. 胚胎可以掏出使用产钳的提示。皮尔斯蜕膜蜕膜与周围的胚胎钳和开放钳撕除。

    图5

    图5

  6. 一旦胚胎被删除,可能仍然附着Reichart的膜以及ectoplacental锥(滋养)。胚胎可以进一步仔细解剖解剖。

    图6
    图6

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Discussion

在小鼠植入后发生4.5 DPC和出生。 ,本质上描述的那样,这夹层的方法可应用于隔离较早或较晚的阶段胚胎在5.5-8.5 DPC。

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Materials

Name Type Company Catalog Number Comments
Micro Dissecting Forceps serrated, straight Tool Roboz Surgical Instruments Co. RS-5130 2 pairs
Micro Dissecting Tweezers Pattern #55 INOX Tool Roboz Surgical Instruments Co. RS-5010 2 pairs
Dulbecco’s Phosphate Buffered Saline (1X) Reagent Invitrogen 14190
Fisherbrand Sterile Polysytrene Petri Dish Tool Fisher Scientific 08-757-12
4% Paraformaldehyde in PBS Reagent
Straight Sharp-Pointed Dissecting Scissors Tool Fisher Scientific 08-940
Pregnant female mice Animal
Dissecting Microscope Microscope

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References

  1. Nagy, A., et al. Manipulating the mouse embryo: A laboratory manual third edition. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York. (2003).

Comments

6 Comments

  1. How you set the exactly time of embryos stage?
    thanks.

    Reply
    Posted by: Anonymous
    May 6, 2008 - 12:47 PM
  2. One option for obtaining pre-implantation embryos is to set up natural matings.  To set up matings, females (6 weeks to 4 months of age) are placed with males (one or two females in each cage with one male) overnight.  The morning after mating, the females can be checked for the presence of a copulation plug in the vagina.  The presence of the vaginal plug indicates that the mating occured, but it dŒs not always mean that a pregnancy will result.  The convention for timed pregnancy assumes that fertilization takes place around midnight during a 7pm to 5am dark cycle, then at noon on the following day (i.e., the day on which the vaginal plug is found), the embryos are aged "0.5 dpc (days post coitum)".  At noon on the next day, the embryos are 1.5 dpc, and so on. It is important to check for plugs early in the morning because they fall out or are no longer detectable ~1² hours after mating, or sometimes earlier.  It is important to note that embryos within a litter can vary with regard to stage of development.  Therefore, morphological criteria are used when more precise embryo staging is required such as the "Theiler Staging Criteria for Mouse Embryonic Development". 

    Reply
    Posted by: Anonymous
    May 19, 2008 - 7:59 PM
  3. I just want to thank you for posting this video, it was extremely useful.  I am a high school student in Wisconsin and I am doing research in stem cells and I'm trying to perfect my dissection method.  I love learning new methods and new information on this subject.  Thank you again. Sara

    Reply
    Posted by: Anonymous
    May 6, 2008 - 6:35 PM
  4. HI! How do you dissect the embryo out of Reichardt's membrane and ectoplacental cone (passage Fig 6A to B)? Thanks - Delphine

    Reply
    Posted by: Anonymous
    June 19, 2009 - 11:36 AM
  5. Do you know if there are any workshops for isolating e5-e9 mouse embryos? You're video was very helpful.

    Reply
    Posted by: Monika L.
    January 14, 2010 - 6:12 PM
  6. I have the same question. How do you dissect the embryo out of Reichardt's membrane and ectoplacental cone (passage Fig 6A to B)? Are their any tricks? Thanks

    Reply
    Posted by: Zhuoqing F.
    December 27, 2012 - 5:34 AM

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