An efficient and safe strategy for germ cell-specific automatic excision of foreign DNA in F1 hybrid transgenic silkworms.

The safety of transgenic technology is a major obstacle in the popularization and use of transgenic silkworms and their products. In sericulture, only the first filial generation (F1 ) hybrid eggs produced by cross-breeding Japanese and Chinese original strains are usually used for the large-scale breeding of silkworms, but this may result in uncontrolled transgene dispersal during the popularization and application of the F1 hybrid transgenic eggs. To address this issue, we developed a safe and efficient strategy using the GAL4/Upstream activating sequence (UAS) system, the FLP/flippase recognition target (FRT) system, and the gonad-specific expression gene promoters (RSHP1p and Nanosp) for the germ cell-specific automatic excision of foreign DNA in the F1 hybrid transgenic silkworms. We established 2 types of activator strains, R1p::GAL4-Gr and Nsp::GAL4-Gr, containing the testis-specific GAL4 gene expression cassettes driven by RSHP1p or Nanosp, respectively, and 1 type of effector strain, UAS::FLP-Rg, containing the UAS-linked FLP gene expression cassette. The FLP recombinase-mediated sperm-specific complete excision of FRT-flanked target DNA in the F1 double-transgenic silkworms resulting from the hybridization of R1p::GAL4-Gr and UAS::FLP-Rg was 100%, whereas the complete excision efficiency resulting from the hybridization of Nsp::GAL4-Gr and UAS::FLP-Rg ranged from 13.73% to 80.3%. Additionally, we identified a gene, sw11114, that is expressed in both testis and ovary of Bombyx mori, and can be used to establish novel gonad-specific expression systems in transgenic silkworms. This strategy has the potential to fundamentally solve the safety issue in the production of F1 transgenic silkworm eggs and provides an important reference for the safety of transgenic technology in other insect species.

Scavenger Receptor Class B Type I Deficiency Induces Iron Overload and Ferroptosis in Renal Tubular Epithelial Cells via Hypoxia-Inducible Factor-1α/Transferrin Receptor 1 Signaling Pathway.

Aims: Scavenger receptor class B type I (SRBI) promotes cell cholesterol efflux and the clearance of plasma cholesterol. Thus, SRBI deficiency causes abnormal cholesterol metabolism and hyperlipidemia. Studies have suggested that ferroptosis is involved in lipotoxicity; however, whether SRBI deficiency could induce ferroptosis remains to be investigated. Results: We knocked down or knocked out SRBI in renal HK-2 cells and C57BL/6 mice to determine the expression levels of ferroptosis-related regulators. Our results demonstrated that SRBI deficiency upregulates transferrin receptor 1 (TFR1) expression and downregulates ferroportin expression, which induces iron overload and subsequent ferroptosis in renal tubular epithelial cells. TFR1 is known to be regulated by hypoxia-inducible factor-1α (HIF-1α). Next, we investigated whether SRBI deletion affected HIF-1α. SRBI deletion upregulated the mRNA and protein expression of HIF-1α, and promoted its translocation to the nucleus. To determine whether HIF-1α plays a key role in SRBI-deficiency-induced ferroptosis, we used HIF-1α inhibitor and siHIF-1α in HK-2 cells, and found that downregulation of HIF-1α prevented SRBI-silencing-induced TFR1 upregulation and iron overload, and eventually reduced ferroptosis. The underlying mechanism of HIF-1α activation was explored next, and the results showed that SRBI knockout or knockdown may upregulate the expression of HIF-1α, and promote HIF-1α translocation from the cytoplasm into the nucleus via the PKC-ß/NF-κB signaling pathway. Innovation and Conclusion: Our study showed, for the first time, that SRBI deficiency induces iron overload and subsequent ferroptosis via the HIF-1α/TFR1 pathway.