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6.5 DPCのマウス胚の解剖

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Massachusetts General Hospital, Harvard Stem Cell Institute, Harvard Medical School

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Cite this Article: 6.5 DPCのマウス胚の解剖

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

Abstract: 6.5 DPCのマウス胚の解剖

哺乳類の胚発生の初期段階での遺伝子発現パターンの解析は、遺伝子の機能、細胞間相互作用とシグナル伝達機構そのガイド胚のパターン形成についての重要な手がかりを提供することができます。しかし、まもなく移植後の脱落膜からマウス胚の解剖は、挑戦的な手順にすることができ、このプロセスの詳細なステップバイステップの資料が不足している。

ここでは、移植後(6.5 DPC)胚が最初に妊娠マウス(膣栓が指定されていた一日0.5 poistの交尾の検出)の子宮を解剖し、その後母体脱落膜から胚を解剖によって分離されている方法を示しています。ライヘルトの膜の郭清は、栄養膜の円錐の除去と同様に記述されています。

Protocol: 6.5 DPCのマウス胚の解剖

雌のマウスからの生殖器官の解剖

  1. 妊娠動物は、圧縮ガスを使用してCO 2窒息により安楽死させる。
  2. 吸収性パッド上に動物の仰臥位に置き、マウスの毛で解剖を汚染の危険性を減らすために70%エタノールに浸けます。
  3. 皮膚をつまんで、通常の手術用はさみで正中線にある小さな横切開を行います。しっかりと切開の上下の皮膚を持ち、腹部を露出する頭部と尾部に向かって離れて皮膚を引っ張る。
  4. 鉗子で腹膜を掴んで、腹腔を露出するカット。
  5. two子宮角、卵管と卵巣:体腔の背側領域での生殖器官を見つけます。

プリミティブストリーク段階(6.5 DPC)解剖

  1. 卵管下子宮を把握し、子宮角を取り出して、子宮間膜に沿って、それがフリーカット。氷冷PBSで切除した子宮を置きます。

    図1

    図1

  2. 子宮角に沿って注入部位との間で切断することにより、各胚を区切ります。

    図2

    図2


  3. 周囲の筋層と包ま脱落膜組織との間に時計メーカーの鉗子をスライドさせて筋肉の子宮内膜をつかみます。筋層バックピール、脱落膜を露出させる。

    図3

    図3

  4. 同封の胚のmidventralまたは遠位端を露出される頂点(脱落膜組織の約1 / 5)で露出脱落膜の一部をオフにクリップ。

    図4

    図4

  5. 胚は、鉗子の先端を使用して砲撃することができます。ピアース離れて脱落膜を引き裂くために胚とオープン鉗子を取り巻くピンセットで脱落。

    図5

    図5

  6. 一度胚が削除される、Reichartの膜はまだだけでなく、栄養膜のコーン(栄養膜)を結合することができる。胚は、慎重に解剖によって、さらに解剖することができます。

    図6
    図6

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Discussion: 6.5 DPCのマウス胚の解剖

マウスのPostimplantationは4.5 DPCと出産の間で発生します。この解剖の方法は、前または後5.5〜8.5 DPCでのステージの胚を分離するために、基本的に前述のように、適用することができます。

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Disclosures: 6.5 DPCのマウス胚の解剖

Materials: 6.5 DPCのマウス胚の解剖

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

References: 6.5 DPCのマウス胚の解剖

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

Ask the Author: 6.5 DPCのマウス胚の解剖

5 Comments

How you set the exactly time of embryos stage?


thanks.

1

Reply

Posted by: RodrigoMay 6, 2008, 12:47 PM

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 does 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". 

1.1

Reply

Posted by: KellyMay 19, 2008, 7:59 PM

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

2

Reply

Posted by: SaraMay 6, 2008, 6:35 PM

HI! How do you dissect the embryo out of Reichardt's membrane and ectoplacental cone (passage Fig 6A to B)? Thanks - Delphine

4

Reply

Posted by: DelphineJune 19, 2009, 11:36 AM

Do you know if there are any workshops for isolating e5-e9 mouse embryos? You're video was very helpful.

6

Reply

Posted by: Monika L.January 14, 2010, 6:12 PM

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

8

Reply

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

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