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 JoVE Biology

Dissection of 6.5 dpc Mouse Embryos

1, 1

1Massachusetts General Hospital, Harvard Stem Cell Institute, Harvard Medical School

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    Summary

    Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

    Date Published: 2/25/2007, Issue 2; doi: 10.3791/160

    Cite this Article

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

    Abstract

    Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

    Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

    Protocol

    Dissection of reproductive organs from a female mouse

    1. Pregnant animals are euthanized by CO2 asphyxiation using compressed gas.
    2. Lay the animal supine on absorbent pad and soak it in 70% ethanol to reduce the risk of contaminating the dissection with mouse hair.
    3. Pinch the skin and make a small lateral incision at the midline with regular surgical scissors. Hold the skin firmly above and below the incision and pull the skin apart toward the head and tail to expose the abdomen.
    4. Grasp the peritoneum with forceps and cut to expose the abdominal cavity.
    5. Locate the reproductive organs in the dorsal region of the body cavity: two uterine horns, the oviduct and the ovaries.

    Primitive Streak-stage (6.5 dpc) Dissection

    1. Remove the uterine horn by grasping the uterus below the oviduct and cut it free along the mesometrium. Place dissected uterus in ice cold PBS.

      Figure 1

      Figure 1

    2. Separate each embryo by cutting between implantation sites along uterine horn.

      Figure 2

      Figure 2


    3. Grasp the muscular uterine lining by sliding watchmaker's forceps between the surrounding muscle layer and enveloped decidua tissue. Peel back the muscle layer, exposing the decidua.

      Figure 3

      Figure 3

    4. Clip off a portion of the exposed decidua at the apex (approximately 1/5 of the decidua tissue) which will expose the midventral or distal tip of the enclosed embryo.

      Figure 4

      Figure 4

    5. The embryo can be shelled out using the tips of forceps. Pierce the decidua with forceps surrounding the embryo and open forceps to tear decidua apart.

      Figure 5

      Figure 5

    6. Once embryo is removed, Reichart's membrane may still be attached as well as the ectoplacental cone (trophoblast). The embryo can be dissected further by careful dissection.

      Figure 6
      Figure 6

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    Discussion

    Postimplantation in the mouse occurs between 4.5 dpc and birth. This dissection method can be applied, essentially as described, to isolate earlier or later stage embryos at 5.5-8.5 dpc.

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    Disclosures

    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

    References

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

    Comments

    6 Comments

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

    Posted by: AnonymousMay 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 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: AnonymousMay 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
    Reply

    Posted by: AnonymousMay 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
    Reply

    Posted by: AnonymousJune 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.
    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
    Reply

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

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