Method for Culture of Early Chick Embryos ex vivo (New Culture)

Summary

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.

Abstract

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Protocol

Part 1: Bench set up A humid chamber is prepared by placing Kimwipe/ddH2O in plastic chamber. A Falcon tube to collect albumin, dishes for culture, rings, watchglass and waste disposal are placed on bench. Pyrex dish is filled with 1.4 l saline (see notes [a]). Part 2: Embryo is explanted in saline Eggs are removed from the incubator after 16 hrs (stage 4). The egg is opene by tapping the shell with forceps. Shell pieces are rem…

Discussion

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.

Materials

Material Name	Tipo	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