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Blood, Vol. 111, Issue 7, 3849-3858, April 1, 2008
Knock-in of an internal tandem duplication mutation into murine FLT3 confers myeloproliferative disease in a mouse model
Blood Li et al.
111: 3849
Supplemental materials for: Li et al
Primers and probe used for genotyping analysis and RT-PCR analysis The following primers were used for PCR and quantitative RT-PCR analyses:
P1F: 5′-CGT CGA GAT CCG GAA CAT AA-3′
P1R: 5′-TCA AGT ACA CTG GCC CTG AA-3′
P2F: 5′-TGC AGA TGA TCC AGG TGA CT-3′
P2R: 5′-CTC TCG GGA ACT CCC ACT TA-3′
P3F: 5′-GTT CAT CAC CCT CCC GAA AG-3′
P3R: 5′-GCT CTG TCT CCC CTT CAT TG-3′
mS16F: 5′-TGT TCC TAC CCC CAA TGT GT-3′
mS16R 5′-TGT GAG GGA GAT GCT CAG TG-3′ Probe A used for Southern blotting analysis was a 658bp fragment of MfeI-XbaI digestion outside of the homologous recombinant region, which yields 6.4 and 8.2 kb fragments for wild-type and ITD alleles, respectively. Antibodies used for flow cytometry The following anti-mouse monoclonal antibodies were used for the staining of cell surface markers:
Alexa Flour 488-conjugated lineage mixture (Invitrogen), which consists of anti-mouse CD3e (clone 500A2), CD11b (Mac-1, clone M1/70.15), CD45R/B220 (clone RA3-6B2), Ly6C/G (Gr-1, clone RB6-8C5) and Ter119 antibodies; FITC-conjugated anti-mouse CD41a (clone MWReg30), CD3 (clone 17A2), CD4 (clone GK1.5), Mac-1 (clone M1/70), NK1.1 (clone PK136), CD45.2 (clone 104) and FcεRIα (clone MAR-1, eBioscience, San Diego, CA); PE-conjugated anti-mouse CD135 (clone A2F10.1), Ter119 (clone Ter119), CD43 (clone S7), CD3 (clone 17A2), CD86 (clone GL1) and CD45.1 (clone A20); PerCP conjugated anti-mouse CD90 (Thy1.1, clone OX-7) and CD8a (clone 53-6.7); PerCP-Cy5.5 conjugated anti-mouse B220 (clone RA3-6B2), Mac-1 (clone M1/70) and CD45.2 (clone 104); PE-Cy5.5 conjugated anti-mouse Ly6A/E (Sca-1, Clone D7, Invitrogen); APC conjugated anti-mouse CD117 (c-KIT, clone 2B8), Gr-1 (clone RB6-8C5), IgM (clone II/41), CD8a (clone 53-6.7) and CD11c (clone HL3) (all except those specifically mentioned are from Becton Dickinson).
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