Windowing Chicken Eggs for Developmental Studies

Published 10/01/2007
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Biology

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Summary

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

Cite this Article

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J. Korn, M., S. Cramer, K. Windowing Chicken Eggs for Developmental Studies. J. Vis. Exp. (8), e306, doi:10.3791/306 (2007).

Abstract

The study of development has been greatly aided by the use of the chick embryo as an experimental model. The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation These experiments have yielded important data on the development of the central and peripheral nervous systems. For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth. The embryo can be examined at successive developmental stages by re-opening the eggshell. While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times. In this method, the egg rests on its side and a small window is cut in the shell. After the experimental procedure, the shell is used to cover the egg for the duration of its development. Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period. This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

Protocol

1. Remove eggs from incubator

  1. Maintain at 37°C with relative humidity set above 60%.
  2. Remove the eggs; turn eggs 90° so that the large base lies horizontal.

2. Swab eggs to sterilize

  1. Saturate a stack of non-sterile gauze with 70% ethanol.
  2. Use two to three pieces to swab up to 5 eggs. Discard when the gauze is soiled.

3. Preparing albumen removal site

  1. Cut and place a 1" x 1" piece of 3M plastic tape just left of the base to protect the area where the albumin will be drawn out.

4. Removal of albumen

  1. Use the point of a pair of scissors to make a small hole in the middle of the tape.
  2. Using a 10 cc syringe with an 18-gauge, 1-inch needle, slowly drill the needle through the hole made by the scissors.
  3. Drive the needle down at a 45°C angle towards the bottom of the egg.
  4. Tilt the needle towards the center and draw up 3 to 4 mL of albumen.

5. Windowing

  1. Cut a 3" x 3" piece of plastic tape and stretch it to fit on the top of the egg. Extend the corners of the square around the rounded ends of the horizontal surface of the eggs, being careful not to pull too hard. Pull the tape so that it is tight against the surface of the eggs with no folds.
  2. Using a pair of sharp-straight 4" dissection scissors, twist a hole into the bottom center of the area where the tape was placed. Slowly guide the lower blade of the scissors into the egg being sure to keep the tips up against the inside of the shell. Direct the blade towards the base and slowly begin to cut the shell. Proceed in a counter-clockwise fashion, stopping just before reaching the top center. Remove the scissors and repeat going in the opposite direction until only a small bit of the egg remains attached. Check to be sure the egg is fertilized. Shut the window.

6. Closing, reopening and sealing the egg.

  1. Cut about a 2-3" long by 1/2" wide plastic tape and shut the window so it fits back into the hole that was cut. Take another 1 x 1" piece of tape and seal the hole from which the egg was drained. Use a pair of forceps to reopen the egg to do any manipulations. When you're ready to return the eggs to the incubator, cut a piece of tape that is large enough to seal the window and cover the entire horizontal surface of the egg.

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Discussion

This method allows for long survival times following experimental manipulations. For example, dye-labeling studies allowed survival to E10 or later in a fate mapping study (1). In addition, it has been used for in ovo electroporation studies that provide alteration of gene expression levels and required development to proceed to later developmental stages (2-6). This windowing technique can be used in combination with any procedure that requires survival after manipulations to the embryo.

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Materials

Name Type Company Catalog Number Comments
Rotating incubator Profi Maintain at 37° Degrees with relative humidity set above 60%
70 % EtOH
gauze
3M plastic tape
10cc syringe 1/egg
18G needles 1½ inch needle, 1/syringe
Dissection scissors 4" sharp straight blades
Fertilized Eggs

DOWNLOAD MATERIALS LIST

References

  1. Cramer, K. S., Fraser, S. E., Rubel, E. W. Dev Biol. 224, 138-151 (2000).
  2. Cramer, K. S., Bermingham-McDonogh, O., Krull, C. E., Rubel, E. W. Dev Biol. 269, 26-35 (2004).
  3. Cramer, K. S., Cerretti, D. P., Siddiqui, S. A. Dev Biol. 295, 76-89 (2006).
  4. Huffman, K. J., Cramer, K. S. Dev Neurobiol. 67, 1655-1668 (2007).
  5. Krull, C. E. Dev Dyn. 229, 433-439 (2004).
  6. Gerlach-Bank, L. M., Cleveland, A. R., Barald, K. F. Dev Dyn. 229, 219-230 (2004).

Comments

12 Comments

  1. Place a 1mm x 1mm piece of 3M plastic tape just left of the base to protect the area where the albumin will be drawn out.Not mm, but cm... It is not a typo, the error is repeated several times in the video...

    Reply
    Posted by: Anonymous
    April 23, 2008 - 8:44 AM
  2. The appropriate measurements are included in the protocol.  As a rule of thumb, cut the tape to a length which will successfully complete the task. 

    Reply
    Posted by: Anonymous
    June 8, 2008 - 8:18 PM
  3. what is the age of the egg when placed in the incubator? pls reply asap. we'll do this protocol in the lab. thanks so much....

    Reply
    Posted by: Anonymous
    June 22, 2008 - 7:28 AM
  4. When the eggs arrive, they are considered to be E0.  Some procotols suggest placing them at 4C, we leave them at room temperature.  Either way, when they are placed in the incubator they are E0.  This means that ²4 hours after you place them in the incubator, they are E1.  Most of our manipulations are done at E²-3 and the eggs are immedialtey placed back in the incubator.

    Reply
    Posted by: Anonymous
    June 22, 2008 - 1:28 PM
  5. Hi, Thanks for the wonderfull information.But I have a small query Is this called [edited] The Chick Chorio Allantois Membrane assay (CAM),if not will you please explain it if you have some idea of the same. Thanks   S.S.Raza MDC,Berlin syedshadab.raza@mdc-berlin.de

    Reply
    Posted by: Syed R.
    March 22, 2009 - 7:39 AM
  6. Hello, Thank you for your comments.  The CAM assay is not directly related to the preparations in the video.  CAM is used specifically for studying angiogenesis and I believe is usually considered an in vitro assay.  This preparation will allow for access to the embryo and is often used for in ovo manipulations that require the development of the embryo.  Best,
    Matt

    Reply
    Posted by: Anonymous
    March 22, 2009 - 11:04 PM
  7. What's reason for removel the albument?Must we do it?What's the percentage of eggs survivalling to E10 after windowing?

    Reply
    Posted by: Anonymous
    June 15, 2011 - 11:11 PM
  8. It is necessary to remove the albumin to allow the embryo to accessed and manipulated once the egg is windowed. If you do not pull out the suggested amount of albumin, the embryo will remain out of reach. Depending on the manipulations, eggs will have a fair to good chance of surviving to E10. For electroporation or bead placement, I'd say it about 60-70% chance the embryo will survive. Good luck.

    Reply
    Posted by: Anonymous
    June 16, 2011 - 2:26 AM
  9. Thank you for your help! We've suceeded in windowing the eggs and they have a good survival.Howerver,we meet another problem.While injecting the india ink ,the eggs will die quickly the next day.How to prepare the ink?And what should we pay attention to when injecting the ink?

    Reply
    Posted by: Anonymous
    July 14, 2011 - 8:05 AM
  10. Hi Camilla,

    I apologize for the delay in responding. The ink is a tricky thing. We went through a period where our survivor rate dropped considerably. After trying several dilutions and filtration, we found that the "new" ink we had purchased was simply not compatible. We reordered Higgins Fountain Pen India Ink, item 46030. Our standard protocol calls for 4% in sterile water. Best of luck.

    Reply
    Posted by: Anonymous
    August 1, 2011 - 1:20 PM
  11. Hi, Thank you very much, This vedio is really helpful.
    Could you please tell me from where I can get that stretchy plastic tape and is it gas permeabile i.e can allow gases exchange

    Reply
    Posted by: Anonymous
    April 16, 2012 - 9:40 AM
  12. Apologies for the delay. My suggestion would be to contact the 3M company directly. The product number is 191CL and ask for stores near you that would carry the product. Depending on your resources, you might want to try a hardware store in the plumbing section. If you cannot find the clear tape, black electrical tape will work as well because it dŒs have some elasticity. Both should allow for normal gas exchange. Good luck!

    Reply
    Posted by: Anonymous
    May 1, 2012 - 7:12 PM

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