Fig 1.
Construction and characterization of PDGF-CChum mice.
The alignment of mouse and human PDGF-CC amino acid sequence is displayed. Red arrows indicate those amino acids that were subjected to point mutations (A). To exchange the mouse with the human PDGFC sequence 6 mutations were introduced into the mouse Pdgfc sequence. K242T, K246R and R299S mutations have been introduced into exon 5. The K318R, N342S and A343T mutations have been introduced into exon 6. Mouse genomic fragments and selected features (such as recombination sites and selection markers) were assembled into a targeting vector together with the 6 point mutations. Positive selection markers have been flanked by FRT (Neomycin resistance—NeoR) and F3 (Puromycin resistance- PuroR) sites and have been inserted into intron 4 and intron 5, respectively. The targeting vector was subsequently transfected into the TaconicArtemis C57BL/6N Tac embryonic stem cell (ES) line. The constitutive humanized allele was obtained after in vitro Flp-mediated removal of the selection marker. This allele expressed the mutated PDGF-CC K242T, K246R, R299S, K318R, N342S, A343T protein (B). Weight curve between 8 and 16 weeks of age in male (C) and female (D) PDGF-CChum. Values in C-D represent mean +/- SEM (n = 10 for each genotype and gender) Distribution of genotypes and gender at weaning from heterozygous breedings (n = 57 litters). PDGF-CChum show Mendelian distribution of genotypes and even distribution of sexes (E). Comparison of the body size between PDGFCChum, PDGF-CC-/- and wt littermate (F).
Fig 2.
Humanized Pdgfc transcripts and PDGF-CC protein are expressed in PDGF-CChum mice.
Primers specific for the mutated Pdgfc detect Pdgfc in PDGF-CChum but not in wt mice (A). Primers specific for mouse Pdgfc detect Pdgfc in wt but not in PDGF-CChum mice (B). Pdgfc (C) and Pdgfrα (D) transcript levels are similar in PDGF-CChum and wt mice (n = 3 for PDGF-CChum and wt mice, respectively). Values in A-D represent mean +/- SEM. Immunostaining with mAB 6B3 in the PDGF-CChum mice (E-H). shows PDGF-CC expression in neurons (E) and blood vessels (E and F) in the brain. mAb 6B3 detects PDGF-CC in the spleen (G) and kidney (H). scale bars: 30 u μm (E-G) and 10 μm (H).
Fig 3.
Female and male PDGFCChum were intraperitoneally injected weekly with 30 mg/kg 6B3 (n = 5 for each gender) or BM4 (n = 5 for each gender) for 15 weeks. Both male (A) and female (B) PDGF-CChum mice treated with 6B3 did not show any weight alterations compared to BM4 treated mice. Organs were harvested and the wet weight of the displayed organs did not differ between the 2 treatment groups (n = 5 for each treatment group) (C). Urine was harvested after 15 weeks and the parameters indicated in the table analyzed (n = 5 for each treatment group) For the ph value SEM is indicated in brackets. (D). Values in A-C represent mean +/- SEM.
Table 1.
Serum parameters after 15 weeks of 6B3 or BM4 injection.
Table 2.
Hematology parameters after 15 weeks injections with 6B3 or BM4.
Fig 4.
Long-term 6B3 treatment does not induce pathology of the major organs.
Female and male PDGFCChum were intraperitoneally injected weekly with 30 mg/kg 6B3 (n = 5 for each gender) or BM4 (n = 5 for each gender) for 15 weeks. Tissue sections of brain (A), kidney (B), liver (C), lung (D), pancreas (E), skin (F), spleen (G) and uterus (H) were stained with HE and their morphology and pathology evaluated. Representative images of female mice are shown. H: Hippocampus, E: Ependymal cells; G: Glomerulus; RC:; T: Trachea, AD: Alveolar duct, A: Aveoli; C: Centroacinar cells, E: Exocrine duct; F: B cell-rich follicle, T: T cell-rich zone.