|
|
Blood, Vol. 113, Issue 14, 3352-3362, April 2, 2009
Novel roles for erythroid Ankyrin-1 revealed through an ENU-induced null mouse mutant
Blood Rank et al.
113: 3352
Supplemental materials for: Rank et al
N-ethyl-N-nitrosourea (ENU) mutagenesis
Male BALB/c Mpl−∕− mice were injected intraperitoneally with three weekly 100 mg/kg doses of the chemical mutagen, ENU (Sigma). Three months later, treated mice were mated with untreated BALB/c Mpl−∕− females to yield first-generation (G1) progeny. At 7 weeks of age, blood from G1 mice was collected from the retro-orbital plexus into tubes containing potassium EDTA (Sarstedt), and the full blood count in the peripheral blood was determined using an Advia 120 automated hematological analyzer (Bayer). The affected G1 mice were crossed with wild type BALB/c animals to segregate the Mpl−∕− locus out of the line. Mice with a low MCV that were wild type at the Mpl locus were identified, and these animals were used for all subsequent experiments. Measurement of reticulocyte percentage
The corrected reticulocyte percentage was obtained from a manual count of new methylene blue stained blood films from three RBC2/RBC2, and three control RBC2/+ and wild type mice. Reticulocytes were counted using the ×100 objective. For wt and RBC2/+ samples at least 100 reticulocytes, and for RBC2/RBC2 samples 200 reticulocytes were counted for each sample. The total number of red cells in the respective fields was also counted, and the reticulocyte percentage calculated.
Files in this Data Supplement:
- Figure S1 (JPG, 165 KB)
-
(A) ENU mutagenesis strategy mice to identify dominant mutations that affected erythroid production and maturation. (B) MCV/RBC distribution in progeny from RBC2/+ x BALB/c matings. (C) New methylene blue stained peripheral blood film for 5-week-old wt, RBC2/+ and RBC2/RBC2 mice for analysis of percentage reticulocytes. The bottom right panel shows a magnified view of the marked rectangle in the bottom left panel.
|
|
