Figure 3.

FIGE analysis of TRF length of bone marrow cells during serial HSC transplantation. (A) Southern blot analysis of TRF length by FIGE. In this sample analysis, all primary recipients and two secondary recipients, represented by the rightmost two lanes, were reconstituted with 100 pure HSCs. The other two secondary recipients were reconstituted with 2 × 10⁶ bone marrow cells. Whole bone marrow cells were isolated from adult BA mice and recipient mice and digested with restriction enzymes as described (see Materials and Methods). 0.5 μg of each digested DNA sample was resolved in a 0.75% agarose gel by FIGE (pulse conditions, 0.8 V forward and 0.4 V reverse for 12 h). The bone marrow sample of the donor mouse in each round of transplantation is indicated by an arrow (↓) at the top. The gel was dried and the DNA hybridized to a ³²P-end labeled telomeric oligomer as described (see Materials and Methods, and reference 23). Sizes of molecular weight standards (in kb) are shown on the left. (B) Measurement of mean TRF length during serial transplantation. The mean TRF length was calculated as described previously (references 23, 29) for a total of 9 sibling adult BA mice, 11 primary recipients, and 10 secondary recipients and averaged for all experiments. Error bars (standard deviation) and P values (Student's t test) are shown.