GreenFluorescentProteinasanIndicatorofTransfectioninChickenEmbryos

Green fluorescent protein (GFP) is responsible for the bioluminescence of the Pacific Northwest jellyfish, Aequorea victoria. In A.victoria, the 27-kDa protein absorbs blue light from a photoprotein that is activated by calcium and emits green light (2). GFP was isolated from A. victoria and the cDNA was originally cloned in 1992 by Douglas Prasher (1). Genetic manipulations of GFP has made it possible for this protein to be expressed in any organism or cell type(2). GFP expression can be readily detected in a specimen using an ultra violet light source. Because GFP expression can be detected in any organism or cell type, this protein has become a very useful transgenetic tool. Some applications of GFP include: determining transcriptional activity of a gene, cell lineage analyze, development of cell and tissue specific markers, and tracing the pathways and development of pathogens and diseases(2). Embryo transfection is an useful tool for studying molecular signaling as well as cell lineages. For example, transfection techniques can be used to supress signaling by introducing antisense nucleotides into the embryo or ectopic expression can be studied by transfecting the embryo with a retrovirus containing a DNA/cDNA insert of choice. Also, dominant/negative genes can be introduced to study the effect of signal interference on cells.

This basic protocol is concerned with the application of GFP as an indicator of transfection in chicken embryos. Lipofectin® Reagent (Life Technologies, catalog no. 18292-011) will be used in an attempt to transfect 2-3 day chicken embryos with a GFP retovirus. This patented lipid reagent binds DNA to form a complex which binds cells and allows for the subsequent uptake and expression of DNA. Although this reagent has been used to transfect individual cell types (fibroblast and blastocyst) of the chicken embryo, it has not been used for entire embryo transfections. Successful DNA uptake will be visualized as fluorescent green granules when the embryo is observed under a fluorescent microscope. More advanced GFP techniques, such as using GFP as a gene reporter in chicken embryos, may be employed using a variation of this protocol once it is evident that this basic transfection procedure is suitable.

Protocol

1) Remove 2-3 day chick embryos using the ring explant technique and store them in sterile Howard's balanced salt solution.

2) Dilute 20µl of Lipofectin® Reagent (Life Technologies, catalog no. 18292-011) into 200µl OptiMem® serum-free medium (Life Technologies) and allow it to stand at room temperature fo 30-45 minutes (Sol. A)

3) For each transfection, dilute 50 µg GFP DNA in 100µl of OptiMem® (Life Technologies) serum-free medium in a sterile microfuge tube (Solution B).

4) Combine Solutions A and B, stir gently and incubate at room temperature for 10-15 minutes to make Solution C.

6) Wash embryos with 2 ml serum free medium.

7) Add 1 ml OptiMem® serum-free medium to the dish containing Solution C. Mix gently and pour over embryo.

8) Incubate the cells for 8 hours in a 5% CO2 incubator at 37ºC.

9) Transfer embryos to albumin agar plates and incubate for 48-72 hours more in a 37º C incubator at 37ºC.

10) Observe embryos using fluorescent microscope 48-72 hours after transfection.

Result

The uptake of green flourescent protein using Lipofecin® was succesful as indicated by the green granules observed using flourescent microscopy. As seen in Figure 2 a anb b, GFP was expressed most noticabley adjacent to the nueral tube in the somite region. The microscope images were imported into Scion Image. The appearance of flourescent granuales confirms that the retrovirus was able to transfect the chicken embryo and maintain transcriptional and translational integrity.

Figure 2. Gfp expression. Embryonic cells were transfected with retroviral Gfp. The cells were successfully transformed as indicated by fluorescing granules which appear light gray to white in the figures above. Images were taken with an attachable black and white microscope camera and imported into Adobe Photoshop for labeling. a) The entire chicken embryo after GFP transfection at (4X). b) A treated embryo exhibits GFP in regions adjacent to the neural tube and in the somites (10X).