This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.
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
Problem | Cause | Remedy |
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.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
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 | M1000 | |
Pyrex dish (2) | Tool | |||
Watchmaker’s glass 50mm | Tool | VWR | 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 | 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 | 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 |