. | . | . | Number of reaggregates . | Number of recipients . | Number of repopulated recipients (% chimerism) . | . | |
---|---|---|---|---|---|---|---|
E10 stage . | AGM marker . | Gut marker . | . | . | AGM marker . | Gut marker . | |
Early | IacZ | Ln72 | 15 | 5 | 1 (43%) | 0 | |
Early | GFP | Ln72 | 6 | 2 | 0 | 0 | |
Early | Ln72 | GFP | 6 | 2 | 0 | 0 | |
Early | Ln72 | IacZ | 9 | 3 | 3 (45%, 47%, 4%) | 0 | |
Late | IacZ | Ln72 | 6 | 3 | 0 | 0 | |
Late | GFP | Ln72 | 2 | 1 | 0 | 0 | |
Late | Ln72 | GFP | 2 | 1 | 1 (6%) | 0 | |
Late | Ln72 | IacZ | 6 | 3 | 3 (98%, 99%*, 100%†) | 0 |
. | . | . | Number of reaggregates . | Number of recipients . | Number of repopulated recipients (% chimerism) . | . | |
---|---|---|---|---|---|---|---|
E10 stage . | AGM marker . | Gut marker . | . | . | AGM marker . | Gut marker . | |
Early | IacZ | Ln72 | 15 | 5 | 1 (43%) | 0 | |
Early | GFP | Ln72 | 6 | 2 | 0 | 0 | |
Early | Ln72 | GFP | 6 | 2 | 0 | 0 | |
Early | Ln72 | IacZ | 9 | 3 | 3 (45%, 47%, 4%) | 0 | |
Late | IacZ | Ln72 | 6 | 3 | 0 | 0 | |
Late | GFP | Ln72 | 2 | 1 | 0 | 0 | |
Late | Ln72 | GFP | 2 | 1 | 1 (6%) | 0 | |
Late | Ln72 | IacZ | 6 | 3 | 3 (98%, 99%*, 100%†) | 0 |
Secondary transplantation . | . | . | Number of BM cells/recipient . | Number of recipients . | Number of repopulated recipients (% chimerism) . | . | |
---|---|---|---|---|---|---|---|
. | AGM marker . | Gut marker . | . | . | AGM marker . | Gut marker . | |
*BM | Ln72 | IacZ | 2×106 | 5 | 5 (99%, 100%, 100%, 29%, 100%) | 0 | |
†BM | Ln72 | IacZ | 2×106 | 5 | 5 (68%, 53%, 83%, 58%, 60%) | 0 |
Secondary transplantation . | . | . | Number of BM cells/recipient . | Number of recipients . | Number of repopulated recipients (% chimerism) . | . | |
---|---|---|---|---|---|---|---|
. | AGM marker . | Gut marker . | . | . | AGM marker . | Gut marker . | |
*BM | Ln72 | IacZ | 2×106 | 5 | 5 (99%, 100%, 100%, 29%, 100%) | 0 | |
†BM | Ln72 | IacZ | 2×106 | 5 | 5 (68%, 53%, 83%, 58%, 60%) | 0 |
Cells from E10 tissues from IacZ, Ln72 and GFP transgenic embryos were made into a chimeric reaggregate and cultured for 3 days. Harvested reaggregate cells were transplanted at a dose of 3 reaggregates(early E10) and 2 reaggregates (late E10) per adult irradiated recipient. At>4 months after transplantation, peripheral blood DNA PCR was performed with IacZ-, Ln72- and/or GFP-specific primers. Percentage donor contribution was determined by comparison of the specific signal that was normalized for DNA content (myogenin control) with serial dilutions (100, 60, 30, 10, 6, 1 and 0%) of control transgenic DNA. n=3.
*,†Individual primary recipient mice repopulated with cells derived from late E10 AGM (Ln72)/gut (IacZ)reaggregates. At the time of sacrifice, hematopoietic tissues were collected and checked by PCR for Ln72 and IacZ markers. Only Ln72 contribution was detected. Bone marrow (BM) of these two primary recipients was used for transplantation into five secondary recipients. At>4 months after transplantation, peripheral blood DNA PCR was performed with IacZ- and Ln72-specific primers.