Embryo Transplantation

1. When dissecting embryos for whole embryo culture it is important to avoid tearing the placenta or damaging major blood vessels in the yolk sac, as this can compromise oxygen

Whole Embryo Culture

Fig. 1. Schematic representation of tissue graft transplantation under kidney capsule, following exposure of the kidney from the abdominal cavity. (A) Grasp the kidney capsule with a pair of fine forceps H and use a second pair P to make a small hole in the membrane. Lift the holding forceps H gently to make a space between the capsule and the surface of the kidney. (B) Bring in the micropipet MP containing the grafts and place into the hole created by the raised forceps H. Blow to release the grafts, stopping before the air bubble reaches the end of the pipet. (C) Remove the micropipet MP, maintaining a positive pressure to ensure the grafts are not taken back up into the pipet. Release the holding forceps H to secure the grafts under the capsule. (D) Kidney recovered 10 d after grafting. The transplanted tissue has increased substantially in size, and can be removed completely from the kidney for analysis.

Fig. 1. Schematic representation of tissue graft transplantation under kidney capsule, following exposure of the kidney from the abdominal cavity. (A) Grasp the kidney capsule with a pair of fine forceps H and use a second pair P to make a small hole in the membrane. Lift the holding forceps H gently to make a space between the capsule and the surface of the kidney. (B) Bring in the micropipet MP containing the grafts and place into the hole created by the raised forceps H. Blow to release the grafts, stopping before the air bubble reaches the end of the pipet. (C) Remove the micropipet MP, maintaining a positive pressure to ensure the grafts are not taken back up into the pipet. Release the holding forceps H to secure the grafts under the capsule. (D) Kidney recovered 10 d after grafting. The transplanted tissue has increased substantially in size, and can be removed completely from the kidney for analysis.

and nutrient flow to the embryo. It is also important to remove tissue debris from the culture medium as this may affect embryo survival.

2. The whole embryo culture system may be utilized in many experiments. For example, it has been used successfully to allow embryonic development after injections of deoxyri-bonucleic acid or morpholino oligonucleotides into the third pharyngeal pouch at E10.5 (Gordon J., Manley, N. R., and Blackburn, C. C., manuscript in preparation) and in bead implant experiments (Moore-Scott, B. A., unpublished data).

3. The maximum time that an E10.5 mouse embryo will survive, grow and develop under the whole embryo culture conditions described is 30 h. This may be caused in part by poor placental development, or to the absence of an as-yet unidentified serum component (4), both of which lead to a compromised nutrient supply.

4. The kidney capsule is well established as providing a permissive environment for growth and development of other organs and tissues (6,7). With respect to the study of thymus development, thymi or cells from any stage may be transplanted under the kidney capsule (8-12), and tissue manipulation such as cell labeling may be performed prior to the transplant. Thus, the technique could be used to extend an analysis that a whole embryo culture-based technique only permits to a limited point.

5. For the isolation of tissues for grafting, the enzyme digestion time is crucial: too long and the tissue will be destroyed, but not long enough and the layers will not separate easily. It is recommended to test an explant after 10 to 15 min and place it back into the enzyme solution if digestion was insufficient. Furthermore, it is important to wash the tissues well after this treatment, as the serum in the medium will inactivate the enzymes and therefore halt the digestion process.

6. It essential to handle the tissue delicately throughout the endoderm dissection. Alternative tools for the dissection include a very fine glass needle or tungsten needle in place of the eyelash tool. The order of dissection is not crucial and may be altered slightly to suit personal preference.

7. When grafting tissue under the kidney capsule, it is important to carry out the transplant as soon as possible after the initial dissection, as the tissue will start to disintegrate even in medium.

8. Alternative methods of anesthesia may be used to perform the grafting experiments. For example, when performing multiple grafts, a stronger dose or alternative substance may be required to allow the operating time to be increased.

9. For tissue transfer, use a minimum volume of liquid in the pipet to avoid flooding of the space under the kidney capsule and loss of the graft. It is also important to push the micropipet at least 5 mm into the space created by the raised forceps to ensure the grafts will remain in place. For grafting larger tissues, forceps may be more suitable than a mouth pipet for placing the grafts under the capsule (a wider diameter pipet will require a larger entry hole and therefore cause more damage). The procedure is more efficient if performed using a dissecting microscope to monitor placement of the tissue fragments.

10. If the tissues or cells being grafted are particularly small, it may be useful to mark the graft site with a small piece of nitrocellulose filter. This should be carefully inserted into the opening in the capsule after the tissue transfer.

11. We routinely use pouch endoderm from two embryos in a single graft. Transplanting tissue from multiple embryos together in a single graft may improve graft recovery. It is theoretically possible to perform grafts from individual embryos, although the survival rate may be reduced. This could be useful if using transgenic or knockout embryos for grafting, where the genotypes of the embryos may not be known before the transplant.

12. Grafts should be sufficiently developed after 10 d to be used for histological or immuno-histochemical analysis. For analysis of T-cells in lymph nodes, grafts should be left for a minimum of 2 to 3 wk.

13. For thymus studies, an important extension of the kidney capsule grafting technique involves the use of nude mice as recipients. This allows for a functional analysis, as the host lymph nodes can be assessed for the presence of peripheral T-cells, which will be present only when a fully functional thymus has developed from the grafted tissue (11).

Optimum results will be obtained using recipient mice syngeneic with the transplanted tissue, to prevent rejection of the graft, even if using nude mouse recipients.

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