hHBB-IVS2-654

Fig.1 Detection of HBB expression in blood by RT-PCR. 

Wild type: only one band at 123 bp with primers F/R(mGapdh) and no band observed by primers for human HBB.

Heterozygous: one band at 303 bp with primers F1/R1(hHBB), one band at 233 bp with primers F1/R2(hHBB)and one band at 123 bp with primers F/R(mGapdh). 

Abbr. M, DNA marker; HE, heterozygous; WT, wild type.

Fig.2 Sequencing analysis of HBB DNA in hHBB-IVS2-654 knock-in mice. Sequencing data confirmed the presence of C to T mutation in the hHBB-IVS2-654 mice. 

Fig.3 Sequencing analysis of spliced HBB mRNA in hHBB-IVS2-654 knock-in mice. Consistent with previous study, sequencing results revealed aberrantly spliced HBB mRNA in hHBB-IVS2-654 knock-in mice. Specifically, these transcripts contained additional sequences corresponding to nucleotides 580–652 of intron 2, which were not present in correctly spliced human HBB mRNA sequence in theory.  

Abbr. HE, heterozygous.

Fig.4 Analysis of spleen and heart coefficients. The tissue coefficient was calculated as (tissue coefficient ‰ = tissue mass/body mass × 1000‰) (n=3/group, 8-week-old, male, Mean ± SEM. T-test, *p < 0.05, ***p < 0.001). As previous study reported, hHBB-IVS2-654 mice showed significant splenomegaly and cardiac hypertrophy caused by hemolysis-derived inflammatory swelling and anemia-derived compensatory hypertrophy.

Abbr. WT, wild type; HE, heterozygous.

Fig.5  Microscopic observation of Wright–Giemsa-stained peripheral blood smears (400x). Red arrows showed target cells and green arrows showed anisocytosis and poikilocytosis. The pictures were scanned with the Leica APERIO VERSA microscope (in-house) (n=3/group, 8-week-old, male). The results showed that hHBB-IVS2-654 mice had severe anisocytosis, poikilocytosis and target cells compared to WT mice. 

Abbr. WT, wild type; HE, heterozygous.

Table 1. Analysis of red blood cells index from the blood routine data of hHBB-IVS2-654 knock-in mice. The changes in HGB, HCT, MCV, MCH, and RDW-CV observed here mirror those documented in hHBB-IVS2-654 mice. Notably, the MCHC index was elevated in reported hHBB-IVS2-654 mice, whereas it is typically reduced in β-thalassemia patients.

Abbr. WT, wild type; HE, heterozygous.

Fig.6 Blood smears analysis. Blood was collect from 12-week-old wild-type mice (1 male and 2 females) and heterozygous hHBB-IVS2-654 mice (1 male and 2 females) for preparation of blood smears and analysis of blood cell morphology.

Conclusion for blood smears analysis:

In wild-type mice: 

1. Mature erythrocytes showed normal morphology and size without other abnormalities; 

2. Leukocyte counts were normal; 

3. Platelet aggregation showed no abnormalities.

In heterozygous hHBB-IVS2-654 mice: 

1. Blood smears revealed a significant increase in target cells, accompanied by anisocytosis, poikilocytosis, and hypochromia (suggestive of thalassemia); 

2. Leukocyte counts were normal, with decreased granulocytes and increased lymphocytes. Small lymphocytes account for a relatively large proportion; 

3. Minor platelet aggregation was observed. 

Remark: Anatomical examination of heterozygous mice revealed splenomegaly.

Fig.7 Prussian blue staining in heterozygous hHBB-IVS2-654 mice. Tissues were collect from 12-week-old wild-type mice (1 male and 2 females) and heterozygous hHBB-IVS2-654 mice (1 male and 2 females) for Prussian blue staining. Iron deposition was observed in the liver, kidney, and spleen of heterozygous hHBB-IVS2-654 mice, with the most prominent accumulation in the spleen.