hSERPINA1-E366K(2)

Fig.1 Detection of SERPINA1 expression in liver by RT-PCR. Mouse Serpina1 mRNA (430 bp) was exclusively detectable in wild-type C57BL/6 mice but not in homozygous hSERPINA1-E366K(2) mice. Human SERPINA1 mRNA (313 bp) was exclusively detectable in homozygous hSERPINA1-E366K(2) mice but not in wild-type mice. Liver RNA was extracted from 6-week-old male/female wild-type C57BL/6 mice (n=3 for each gender) and homozygous hSERPINA1-E366K(2) mice (n=2 for each gender), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mRNA primers. 

Abbr. M, marker; HO, homozygous; WT, wild type.

Fig.2 mRNA sequencing of hSERPINA1-E366K(2) knock-in mice. The sequencing data of RT-PCR products confirmed the presence of target mutations in homozygous hSERPINA1-E366K(2) knock-in mice. 

Abbr. HO, homozygous; WT, wild type.

Fig.3 Detection of SERPINA1 expression in liver by qPCR. Mouse Serpina1 mRNA was exclusively detectable in wild-type C57BL/6 mice but not in homozygous hSERPINA1-E366K(2) knock-in mice. Human SERPINA1 mRNA was exclusively detectable in homozygous hSERPINA1-E366K(2) knock-in mice but not in wild-type mice. Furthermore, SERPINA1 mRNA levels were higher in male than in female hSERPINA1-E366K(2) knock-in mice.

Liver RNA were extracted from 6-week-old male/female wild-type C57BL/6 mice (n=3 in each gender) and homozygous hSERPINA1-E366K(2) knock-in mice (n=2 in each gender), then cDNA libraries were synthesized by reverse transcription, followed by qPCR with human SERPINA1, mouse Serpina1 and mouse Gapdh primers. (A) Relative expression represents the human SERPINA1 mRNA level relative to its average expression in male homozygous hSERPINA1-E366K(2) knock-in mice. (B) Relative expression represents the mouse Serpina1 mRNA level relative to its average expression in male wild-type mice.

Abbr. HO, homozygous; WT, wild type.

Fig.4 Detection of human SERPINA1 expression in serum of WT C57BL/6 mice and HO hSERPINA1-E366K(2) mice by ELISA.  Human SERPINA1 was detectable in serum from homozygous hSERPINA1-E366K(2) knock-in mice. Serum hSERPINA1 levels in both female and male hSERPINA1-E366K(2) knock-in mice were drastically lower than the human plasma hSERPINA1 level. Furthermore, SERPINA1 protein levels were higher in male than in female hSERPINA1-E366K(2) knock-in mice. Serum were collected from wild-type C57BL/6 mice (n=3, 6 weeks old) and HO hSERPINA1-E366K(2) mice (n=2, 6 weeks old), and analyzed by ELISA with anti-hSERPINA1 ELISA kit. 

Abbr.  HO, homozygous; WT, wild type; N.D, not detected.

Fig.5 Detection of human SERPINA1 expression in liver of WT C57BL/6 mice and HO hSERPINA1-E366K(2) mice by ELISA. SERPINA1 was detectable in liver from both WT C57BL/6 and homozygous hSERPINA1-E366K(2) knock-in mice, as the ELISA kit was cross-reactived between human and mouse SERPINA1. Furthermore, SERPINA1 protein levels were higher in male than in female hSERPINA1-E366K(2) knock-in mice. Liver homogenates were collected from wild-type C57BL/6 mice (n=3, 6 weeks old) and homozygous hSERPINA1-E366K(2) mice (n=2, 6 weeks old), and analyzed by ELISA with anti-hSERPINA1 ELISA kit. 

Abbr.  HO, homozygous; WT, wild type.

Fig.6 Detection of human SERPINA1 expression in lung of WT C57BL/6 mice and HO hSERPINA1-E366K(2) mice by ELISA. Human SERPINA1 was detectable in lung from homozygous hSERPINA1-E366K(2) knock-in mice. Furthermore, SERPINA1 protein levels were higher in male than in female hSERPINA1-E366K(2) knock-in mice. Lung homogenates were collected from wild-type C57BL/6 mice (n=3, 6 weeks old) and homozygous hSERPINA1-E366K(2) mice (n=2, 6 weeks old), and analyzed by ELISA with anti-hSERPINA1 ELISA kit. 

Abbr.  HO, homozygous; WT, wild type; N.D, not detected.

Fig.7 Biochemical assessment of hepatic function of hSERPINA1-E366K(2) mice. ALT and AST levels showed a trend toward increase in male hSERPINA1-E366K(2) knock-in mice.

6-week-old WT mice and homozygous hSERPINA1-E366K(2) mice were utilized in this study. Serum samples were collected to quantify the levels of (A) alanine transaminase (ALT), (B) aspartate aminotransferase (AST), (C) total protein (TP) and (D) albumin (ALB). (n=2-3, Mean ± SEM). 

Fig.8 Representative histopathological images of lung from WT mice and homozygous hSERPINA1-E366K(2) mice (n=2-3).

In WT mice, no obvious lesions were observed. In hSERPINA1-E366K(2) mice, mild interstitial inflammatory cell infiltration with parenchymal thickening was observed (black arrows); additionally, mice exhibited alveolar fusion and remodeling, with enlarged and coalescent alveolar spaces and destruction of alveolar walls (blue arrows), as well as bronchial epithelial hyperplasia (red arrows).

Fig.9 Lung pathological scores of male and female hSERPINA1-E366K(2) mice (n=2-3, Mean ± SEM). Homozygous hSERPINA1-E366K(2) mice developed significant lung injury, characterized by mild interstitial inflammatory cell infiltration with parenchymal thickening, alveolar fusion and remodeling, with enlarged and coalescent alveolar spaces and destruction of alveolar walls, as well as bronchial epithelial hyperplasia.