|
|
Blood, Vol. 107, Issue 11, 4317-4325, June 1, 2006
Genetic marking of hematopoietic stem and endothelial cells: identification of the Tmtsp gene encoding a novel cell surface protein with the thrombospondin-1 domain
Blood Takayanagi et al.
107: 4317
Supplemental materials for: Shin-ichiro et al
Supplemental Materials and Methods PCR primers for the Tmtsp gene PCR primers for semi-quantitative RT-PCR:
forward primer; 5′-AAATCCTACACTGTGGGGCATCCCA-3′
reverse primer; 5′-CAGCATTCTCTGCTCAGCCTCGGTG-3′ PCR primers for quantitative RT-PCR:
forward primer; 5′-TGGAATGTCCTCAGAGACCTC-3′
reverse primer; 5′-GAACGGGCGTGCTGCATTTG-3′ PCR primers to detect splicing variant:
forward primer; 5′-AGAGAGGAGAACGGTGTTCA-3′
reverse primer; 5′-CTCCTCCAGAGAGTGATGAG-3′ PCR primers to amplify cDNA probe for northern blotting:
forward primer; 5′-AAGGAATGCATGCTAATTCAG-3′
reverse primer; 5′-CCTAGGTCCGTCACCGGCATCC-3′ PCR primers for genotyping:
forward primer for the wild-type allele;
5′-TTCCCGATCAGAATGAAACCCATG-3′
forward primer for the mutated allele;
5′-GTGGATGTGGAATGTGTGCGAGGC-3′
reverse primer for both allele; 5′-AGGCAAAATCCCCAGGATACAGTA-3′ PCR primers to amplify DNA probe for southern blotting:
forward primer for 5′ probe; 5′-TGTCAGCGTTTTGAGGAACC-3′
reverse primer for 5′ probe; 5′-TACAAATGGATCAAAAACGC-3′
forward primer for 3′ probe; 5′-CTGTCTTGGTGTGGCCAAAA-3′
reverse primer for 3′ probe; 5′-CACAGCTTGGTGCTCTGACT-3′ Combination of antibodies
All antibodies used for flow cytometry were purchased from BD Pharmingen except phycoerythrin (PE)-Flk-1 (eBioscience, San Diego, CA, USA), biotin-conjugated LYVE-1, and allophycocyanin (APC)-VE-cadherin (kindly provided by Dr. Ogawa (Kumamoto Univ.)). Fluorescence of Venus was detected at the fluorescein isothiocyanate (FITC)-channel. Combinations of antibodies were as follows. | 1. Hematopoietic cells of adult Tmtsp+/+ mouse. |
|---|
CD34+/-KSL: | Tmtsp-biotin / streptavidin-PE, c-Kit-APC, Sca-1-PE-Cy5.5, CD34-FITC, and purified-Lin- cells (Gr-1, Mac-1, TER119, B220, IL-7R, CD4, CD8; depleted by MACS anti-rat IgG magnetic beads (Mylteni Biotech)) | | Bone marrow: | Tmtsp-biotin / streptavidin-PE, Mac-1-APC, and Gr-1-FITC | | | Tmtsp-biotin / streptavidin-PE, TER119-APC, and NK1.1-FITC | | | Tmtsp-biotin / streptavidin-PE, B220-APC, and IL7-R-FITC | | Spleen: | Tmtsp-biotin / streptavidin-PE, B220-APC, and Thy1.2-FITC | | | Tmtsp-biotin / streptavidin-PE, and NK1.1-FITC | | Thymus: | Tmtsp-biotin / streptavidin-PE, CD4-FITC and CD8-APC | | 2. Endothelial cells of adult Tmtsp+/+ mouse. |
|---|
Liver: | Tmtsp-biotin / streptavidin-PE, CD45-FITC, and CD31-APC | | 3. Hematopoietic cells of adult TmtspVenus/+ mouse |
|---|
| CD34+/-KSL: | Lin-biotin (Gr-1, Mac-1, TER119, B220, IL-7R, CD4, CD8) / streptavidin-TexasRed, c-Kit-APC, Sca-1-PE-Cy5.5, and CD34-PE | | | CMP, MEP, and GMP: Fc R-PE, c-Kit-APC, Sca-1-PE-Cy5.5, CD34-biotin / streptavidin, and purified-Lin- cells (Gr-1, Mac-1, TER119, B220, IL-7R, CD4, CD8; depleted by MACS anti-rat IgG magnetic beads) | | CLP: | Lin-biotin (Gr-1, Mac-1, TER119, B220, CD4, CD8) / streptavidin-TexasRed, c-Kit-APC, Sca-1-PE-Cy5.5, and IL-7R-PE | | Bone marrow: | Gr-1-PE and Mac-1-APC TER119-PE and B220-APC B220-APC and CD43-PE (for immature B-cells) | | Spleen: | NK1.1-PE and B220-APC Thy1.2-PE Thymus: CD4-PE and CD8-APC (for T-cells) CD4-biotin and CD8-biotin / streptavidin-TexasRed, CD25-APC, and CD44-PE (for DN cells) | | 4. Embryoid body and embryo |
|---|
| Cells from EBs were stained with PE-Flk-1 and APC-conjugated antibodies (CD31, CD41, CD45, TER119, Mac-1). Cells from embryo were stained with APC-CD41. |
Files in this Data Supplement:
- Figure S1. Characterization of the mouse Tmtsp gene (JPEG, 181 KB)
-
(A) Multiple alignment of mouse Tmtsp protein with Unc5h3, ICAM-1, and thrombospondin-1. Identical residues are shaded. The asterisks in Ig-like domain indicate cysteine residues to form the disulfide bond. Bars and the dotted line in TSP1 domain indicate residues corresponding to 2 consensus motifs, WXXW and CSVTCG. (B) Genomic structure of mouse Tmtsp. Locations of the 4 protein motifs are shown. (C) Splice site sequences and exon sizes for mouse Tmtsp. All exons are flanked by GT and AG dinucleotides (underlined) consistent with consensus sequences for splice junctions in eukaryotic genes. DNA sequences of exons and introns are shown in uppercase and lowercase, respectively. (D) Tissue distribution of alternative splicing. PCR primers corresponding to the third (forward) and the fifth (reverse) exons were used to amplify both the full-length Tmtsp and its variant form ( TSP1). Alternative splicing was detected in all tissues tested.
- Figure S2. Expression profile of Venus/Tmtsp in hematopoietic cells (JPEG, 150 KB)
-
Expression of Tmtsp in lineage marker—positive cells was determined by flow cytometric analysis by using Venus knock-in mouse at the Tmtsp locus. Results were shown in histograms (x-axis: fluorescence intensity of Venus; y-axis: relative cell number). (A) Bone marrow cells. (B) Spleen. (C) Thymus.
|
|
