The Construction and Characterization of Mitochondrial Ferritin Overexpressing Mice
Xin Li，Peng Yu，Yan-Zhong Chang
Mitochondrial ferritin (FtMt) is a H-ferritin-like protein which localizes to mitochondria. Previous studies have shown that this protein can protect mitochondria from iron-induced oxidative damage, while FtMt overexpression in cultured cells decreases cytosolic iron availability and protects against oxidative damage. To investigate the in vivo role of FtMt, we established FtMt overexpressing mice by pro-nucleus microinjection and examined the characteristics of the animals. We first confirmed that the protein levels of FtMt in the transgenic mice were increased compared to wild-type mice. Interestingly, we found no significant differences in the body weights or organ to body weight ratios between wild type and transgenic mice. To determine the effects of FtMt overexpression on baseline murine iron metabolism and hematological indices, we measured serum, heart, liver, spleen, kidney, testis, and brain iron concentrations, liver hepcidin expression and red blood cell parameters. There were no significant differences between wild type and transgenic mice. In conclusion, our results suggest that FtMt overexpressing mice have no significant defects and the overexpression of FtMt does not affect the regulation of iron metabolism significantly in transgenic mice.C57BL/6 FtMt overexpressing mice were established by pro-nucleus microinjection with the PiggyBac (PB) System by Cyagen Biosciences Inc. (Suzhou, China). A mouse Ftmt-pcDNA3.1(−) construct was established as previously described . Briefly, mouse Ftmt cDNA encoding the full Ftmt open reading frame was amplified by polymerase chain reaction using 5′-AGGGAATTCACCATGGGCCTGTCCTGCTTTTGGTTCTTCTC-3′, and 5′-GGCGGATCCTATTTAAGCGTAATCTGGAACATCGTATGGGTAGTGCTTGCTCTCGCTTCCAA-3′ primers. The PCR product was inserted into the pGEM-T vector to obtain the plasmid containing a 768-bp fragment including the entire Ftmt sequence with a C-terminal hemagglutinin (HA) epitope and an EcoRI site at the 5′ end, and a BamHI site downstream of the stop codon at the 3′ end. The 768-bp fragment was excised and sub-cloned into the EcoRI/BamHI sites of pcDNA3.1(−) to obtain pcDNA3.1(−)-Ftmt  (Figure 1). The linearized and purified plasmid was diluted to 1 to 5 ng/μL before it was loaded into the microinjection needle. Morphologically normal zygotes were selected to perform the microinjection and then cultured in an incubator for 1 h. Next, the embryos were transplanted into pseudo-pregnant female mice where the development of embryo proceeded to term, twenty days following the procedure . The pups were genotyped by PCR and positive male mice were viable (F0). The foreign gene is usually incorporated into only one chromosome, so the founder mouse is heterozygous. Heterozygous offspring (F1) of both sexes were obtained through founder male mouse crosses with wild-type female mice (Figure 1). We were able to obtain the homozygous mice by sib mating of the heterozygous ones. Litters were genotyped to identify male Tg mice to be used in experiments.Mice were kept in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and by approval of the Animal Care and Use Committee of the Hebei Science and Technical Bureau, and the Laboratory Animal Ethical and Welfare Committee (AEWC) of Hebei Normal University (8 March 2015, Number: 2015-003). Mice were housed in stainless steel cages at 21 ± 2 °C and provided free access to food and water. The rooms maintained a 12 h light and 12 h dark cycle . Age-matched wild-type male mice and FtMt overexpressing male mice (5 months old) were used in this study.The primers that we used to identify the genotype of mice were as follows:Transgene PCR primer forward 5′-CCCACTGCTTACTGGCTTATCGAA-3′, and reverse 5′-TACACGTAGGATGCGTAAAGCTC-3′.Internal control PCR primer forward 5′-CAACCACTTACAAGAGACCCGTA-3′, and reverse 5′-GAGCCCTTAGAAATAACGTTCACC-3′. The internal control PCR targets the endogenous mouse Rgs7 (G protein signaling 7) locus, which exerts the role as internal primer to exclude the influence of the mouse itself.The mice were anesthetized with pentobarbital sodium (40 mg/kg) and then were perfused with 0.9% saline. The body weights of the mice were measured before they were killed. The peripheral tissues (heart, liver, spleen, kidney, testis, thymus) were separated and washed in normal saline solution, dried with sterilized filter paper, and then weighed to calculate the organ coefficient. The tissues were later used to measure iron concentration. The brains of the mice were dissected into cerebral cortex, hippocampus, and striatum, and used for western blot and iron concentration analyses.This work was supported by the National Science Foundation of China (31520103908, 31471035 and 31271473).