Table 1. Contribution of the parental... | American Society of Hematology

Table 1.

Contribution of the parental genotype to individual aggregation chimeras and the genetic composition of LTC-IC in bone marrow

Mouse	Coat color	PCR^*				Staining^†		LTC-IC chimerism
		PCR^*				Staining^†		Measured^‡		Predicted^1-153
		BM	Kidney	Heart	Stroma	BM	Spleen	No. LTC-IC	Genotype	Int	Ext	OMM
2	50	30 ± 0.5	35	50	nd	30	44	70	12.0	3.8	30.0	88.8
3	50	33 ± 0.5	41	57	nd	40	54	48	11.3	5.7	40.0	87.5
5	70	67 ± 4.5	76	79	nd	75	76	53	36.2	21.4	75.0	77.8
6	80	63 ± 2.0	60 ± 2	79	30	73	76	130	25.1	19.6	72.8	78.8
8	20	53 ± 2.0	40 ± 0	58	71	68	78	102	21.9	16.2	68.0	80.8
9	20	26	27 ± 1	53	42	30	39	120	13.7	3.8	30.0	88.8
Controls
B6D2 F1		46 ± 3.1
B6 + D2^1-155						44.2 ± 1.9
B6	100	97 ± 1.6				91.1 ± 5.7
D2	0	8 ± 1.6				0.2 ± 0.2

Mouse	Coat color	PCR^*				Staining^†		LTC-IC chimerism
		PCR^*				Staining^†		Measured^‡		Predicted^1-153
		BM	Kidney	Heart	Stroma	BM	Spleen	No. LTC-IC	Genotype	Int	Ext	OMM
2	50	30 ± 0.5	35	50	nd	30	44	70	12.0	3.8	30.0	88.8
3	50	33 ± 0.5	41	57	nd	40	54	48	11.3	5.7	40.0	87.5
5	70	67 ± 4.5	76	79	nd	75	76	53	36.2	21.4	75.0	77.8
6	80	63 ± 2.0	60 ± 2	79	30	73	76	130	25.1	19.6	72.8	78.8
8	20	53 ± 2.0	40 ± 0	58	71	68	78	102	21.9	16.2	68.0	80.8
9	20	26	27 ± 1	53	42	30	39	120	13.7	3.8	30.0	88.8
Controls
B6D2 F1		46 ± 3.1
B6 + D2^1-155						44.2 ± 1.9
B6	100	97 ± 1.6				91.1 ± 5.7
D2	0	8 ± 1.6				0.2 ± 0.2

PCR, polymerase chain reaction; BM, bone marrow; LTC-IC, long-term culture initiating cells; int, intrinsic; ext, extrinsic; OMM, optimal model mixing; nd, not determined.

Mouse: Individual aggregation chimeras (identified by number) were genotyped. All data are expressed as percentage of B6 contribution to the indicated tissues.

Coat color was estimated by visual inspection.

Semiquantitative PCR was performed on DNA extracted from the indicated organs and cultured bone marrow stromal cell cultures. Values for chimeras are from 1 to 2 independent PCR assays. Parental control mice were performed 4 times. Level of detection of PCR analysis: 5%.

†

Immunofluorescence analysis on chimeric bone marrow and spleen was performed once; the corresponding values for the control mice are means from 10 independent experiments. The staining data depict the percent of B6 cells as determined by gating using Lysis software.

‡

Designates the LTC-IC analyzed from each chimeric mouse; the number of microcultures (No. LTC-IC) tested and the proportion of LTC-IC that were of the B6 genotype are given.

F1-153

The extent of chimerism in bone marrow determined by immunofluorescence before the initiation of the cultures was used to calculate the predicted ratio of LTC-IC if all Scfr genes were to act inside stem cells (intrinsic) or if all activity would be found in the environment (extrinsic). Based on the 11-fold higher frequency of LTC-IC in the parental D2 mice, a factor of 11 was used to multiply the D2 frequency in bone marrow to derive the expected autonomous ratio. The expected extrinsic ratio is identical to the ratio of B6 and D2 cells in the bone marrow of each chimera. OMM was used to calculate for each mouse the percent contribution of the intrinsic model that will give the best fit to the percent B6 actually measured in the LTC-IC. Overall, this yielded 83% ± 4.7% intrinsic regulation.

F1-155

A mixture of 50% each of D2 and B6 bone marrow was tested; the level of staining for the D2 genotype was 49.9 ± 1.9%. Staining of D2 bone marrow with the H-2^d-specific mAb was 94.1 ± 3.1% not significantly different (p = 0.17) from the staining of B6 bone marrow with the H-2^b specific mAb.

View Large

This Feature Is Available To Subscribers Only