1Center for Environmental and Genetic Medicine, Institute of Biosciences and Technology - Texas A&M Health Science Center, 2Center for Environmental and Genetic Medicine, Texas A&M University (TAMU)
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Psychoyos, D., Finnell, R. Method for Culture of Early Chick Embryos ex vivo (New Culture). J. Vis. Exp. (20), e903, doi:10.3791/903 (2008).
Part 1: Bench set up
Part 2: Embryo is explanted in saline
Part 3: Embryo is centered on ring
Part 4: The culture is set up under microscope
Part 5: The culture is transferred to incubator
Part 6: Following culture, the embryo is fixed; culture is transferred to incubator
Part 7: Representative Results
Prior to culture, the embryo is at primitive streak stage (HH4). At the end of the culture period, the embryo has developed to HH10 (length 2-3 mm) and is visible in the center of the culture dish. It is possible to label a group of cells with carbocyanine fluorescent DiI just before culture (0h) and follow their movement throughout the culture period. In this case, cells below Hensen's node (HN) were labelled with DiI. These cells are shown to contribute to progressively developing somites (som) and notochord (n).
Part 8: Troubleshooting
|Embryo remains with yolk instead of coming off with membrane (step 2)||Poor egg quality||Request freshly produced eggs; Store eggs at 13°C upon receipt. Incubate eggs the same day as arrival date.|
|Vitelline membrane slide away from watchmaker's glass following placement of ring (step 3)||Albumin remnants||In step 2, make sure that all albumin is removed by pulling it away from membrane with tilted forceps|
|Embryo is inaccessible: lies underneath the membrane (step 4)||Wrong side of membrane is upwards||In step 3, make sure the side of the membrane containing yolk granules is facing upwards. The shiny, polished side should face downwards.|
|Embryo submerged in saline/albumin following culture(step 6)||Saline/albumin left inside ring prior to culture; hole in membrane||In step 5, make sure that all albumin/saline is removed from inside the ring; In step 4, make sure you do not pierce membrane with forceps (use blunt ended forceps)|
|Embryo disintegrated following culture||Bacterial infection||Sterilize all tools and glassware; Use antibiotic/antimycotic|
The New culture method 2 can be used for a wide variety of applications, ranging from grafts of growth factor containing beads 3, to whole mount in situ hybridization and whole mount immunohistochemistry 4. Culture over a 24 hr period enables the continuous monitoring of embryonic development in applications such as time lapse cell movement analysis 5 or monitoring of GFP containing electroporated constructs 6.
This work was supported by the Margaret M. Alkek Foundation to RHF.
|Eggs||Animal||Charles River Laboratories||Premium Fertile|
|Stereomicroscope||Microscope||Leica Microsystems||MZ9.5 or similar|
|Marsh Automatic Incubator||Tool||Lyon||RX|
|Hybridization Incubator||Tool||Robbins Scientific, SciGene||M1000|
|Pyrex dish (2)||Tool|
|Watchmaker’s glass 50mm||Tool||VWR international||66112-060|
|Glass rings||Tool||Physical Plant facility||cut 4 mm thick sections of glass tubing (27 mm outer diam, 25 mm inner diam). Do not fine polish.|
|Curved Forceps (1)||Surgery||Electron Microscopy Sciences||72991-4C|
|Forceps (2)||Surgery||Fine Science Tools||11002-13||blunt ended using sharpening Stone and 100ul mineral oil|
|Sharpening Stone Dan’s Black Arkansas||Surgery||Electron Microscopy Sciences||62082-00|
|Fine scissors||Surgery||Fine Science Tools||14161-10|
|Plastic dishes||Tool||Falcon BD||353001|
|Rubber Bulb||Tool||Electron Microscopy Sciences||70980|
|Pasteur Capillary Pipette||Tool||Electron Microscopy Sciences||70950-12||round edge under flame|
|Microcapillary tube||Surgery||Sigma-Aldrich||P1049-1PAK||Pull using vertical micropipette puller; blunt end with fine forceps|
|Microdissecting knife||Surgery||Fine Science Tools||10056-12||Use to puncture cavities prior to in situ hybridization|
|Minuten pins 0.2mm diam||Surgery||Fine Science Tools||26002-20|
|Sylgard 184 Silicon Elastomer Curing Agent and Base||Reagent||Dow Corning||0001986475||Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C|
|Diethylpyrocarbonate (depc)||Reagent||Acros Organics||10025025||Add 1ml depc to 1l PBS; shake; autoclave|
1. Pannett, P.A. & Compton, C.A. The cultivation of tissues in saline. Lancet 206, 381-384 (1924).
2. New D.T. A new technique for the cultivation of the chick embryo in vitro. J. Embryol. Exp. Morph. 3, 326-331 (1955).
3. Alvarez, I.S., Araujo, M & Nieto, M.A. Neural induction in whole chick embryo cultures by FGF. Dev. Biol. 199, 42-54 (1998).
4. Psychoyos, D. & Stern, C.D. Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo. Development 122, 3263-3273 (1996).
5. Psychoyos, D. & Stern, C.D. Fates and migratory routes of primitive streak cells in the chick embryo. Development 122, 1523-1534 (1996).
6. Voiculescu O., Papanayotou C. & Stern, C.D. Spatially and temporally controlled electroporation of early chick embryos. Nature Protoc. 3, 419-426 (2008).