Transgenic Research最新文献

Incretin hormones, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), play pivotal roles in glucose homeostasis and metabolic regulation. Therapeutic incretin receptor agonists (RAs), such as tirzepatide, are widely used to manage type 2 diabetes and obesity. However, incretin RAs are facing production challenges at present. Therefore, we engineered transgenic (tg) mice to secrete incretin RAs in milk, leveraging mammary gland bioreactors for cost-effective peptide production. The goat beta-casein promoter-driven constructs encoding tirzepatide-derived peptide linked to human IgG4 Fc via a (GGGGS)₃ spacer were used to produce tg mice. Founders tg-1 and tg-5 exhibited mammary-specific expression, yielding 0.8-1.42 g/l recombinant protein exclusively in milk. Progeny nursed by founders showed sustained hypoglycemia (10-39% reduction; p < 0.05) and marked weight loss (14-49%; p < 0.01) compared to wild-type controls, validating the bioactivity of milk-derived GLP-1/GIP RAs. Moreover, tg-5-nursed offspring experienced high mortality post-Day 16, likely due to overdosing. This proof-of-concept demonstrates the mammary gland bioreactor as a viable platform for incretin RAs production, circumventing complex synthesis and enabling scalable biologics manufacturing.

A new line of cedar pollen allergen-accumulating transgenic rice, which accumulated the 7Crp peptide comprised of 7 concatenated major T cell epitopes of cedar pollen allergens, was developed; it improved on the shortcomings of an original transgenic line. The new line has one copy of inserted T-DNA and a substantial expression of the 7Crp peptide, and was named Os7Crp2. Regulatory approval is needed before commercializing this cedar pollen peptide rice as a genetically modified food product in Japan. Therefore Os7Crp2 was evaluated for the criteria required for food safety assessments, under the guidance of Standards for the Safety Assessment of Genetically Modified Foods (Seed Plants) in Japan. No statistically significant differences were found between Os7Crp2 and the control (parental) Dongtokoi cultivar in physicochemical properties, expressed traits, or concentrations of key components, except for the insertion of the T-DNA and expression of the recombinant protein derived from the transgenes. These results suggest that Os7Crp2 is compositionally equivalent to non-transgenic rice.

Molecular breeding and gene function studies in plants require high transformation efficiency. Agrobacterium-mediated transformation has contributed significantly to molecular research in many plants, but is inefficient and inconsistent in rice that do not host Agrobacterium. Transformation efficiency in rice remains low. Therefore, this study aimed to establish a simple and efficient transformation method for rice using Agrobacterium. Two foreign genes (CISP1-GFP and CISP2-GFP) and Agrobacterium strain (EHA105) was used in the experiments. Then, Agrobacterium infection of rice seeds that had absorbed water and germinated under reduced pressure infiltration conditions showed that an average of 14% of the seeds formed after growth (12% with CISP1-GFP and 16% with CISP2-GFP) carried the foreign gene, and it was also confirmed by PCR, Western blot, GFP fluorescence and TAIL-PCR. Since this method does not involve callus formation or re-differentiation of rice plants, no special equipment or complicated operations are required, and transformants can be obtained in only three months. Therefore, this method is expected to simplify rice genetic manipulation and promote molecular breeding of rice.

Immunodeficient mouse models are invaluable tools for preclinical research, particularly for cancer therapies and studies of the human immune system. Notably, strains with combined Prkdc (scid) and Il2rg (null) mutations-such as NOG and NSG mice- are widely used due to their profound immunodeficiency, allowing efficient engraftment of various human cells. However, these models were generated by disrupting the Il2rg gene through replacement with a neomycin resistance (Neo) cassette in embryonic stem cells. Incomplete excision of this cassette can inadvertently alter the expression of neighboring genes, thereby introducing potential confounding variables. In addition, they may still express mutant mRNAs that escape nonsense-mediated decay (NMD) and/or produce truncated proteins with residual activity, potentially compromising the interpretation of experimental outcomes. To address this, we developed the N2G mouse strain (NOD-2-Genes KO) where almost all genomic loci of both Prkdc and Il2rg genes are deleted via CRISPR/Cas9 genome editing. N2G mice exhibited tumor growth comparable to NOG mice following the transplantation with several human cancer cell lines. Moreover, human CD34⁺ cord blood (CB) cells engrafted into N2G mice showed robust reconstitution of human immune cells, especially T cells in peripheral blood, spleen and bone marrow, compared to NSG mice. These results suggest that N2G mice, lacking residual mutant mRNA and the exogenous Neo resistant gene, offer an advanced model for preclinical studies.

Fabry disease is a rare X-linked inherited lysosomal storage disorder caused by a reduction or deficiency in the activity of α-galactosidase A (α-Gal A). The short half-life of α-Gal A necessitates biweekly infusions, thereby imposing significant economic and physical burdens on patients and their families. In this study, a novel long-acting replacement for α-Gal A, termed α-galactosidase A-Fc (α-Gal A-Fc), was designed. Two transgenic founders with an 18.2% transgene rate were obtained to express recombinant human α-Gal A-Fc protein in mouse milk. The α-Gal A-Fc enzyme activity in the milk of high-copy mice were significantly higher than those in low-copy mice and were stably inherited across F1-F3 generations. No significant differences were observed in α-Gal A-Fc concentration or enzymatic activity among high-copy mice of the same generation. During early lactation, the α-Gal A-Fc concentration and enzymatic activity were 2.1-fold and 2.17-fold higher, respectively, compared to late lactation. The expression levels during late lactation did not affect purification efficiency, allowing for the pooling of milk from high-copy mice throughout the entire lactation period for protein purification. The elimination half-life of the purified α-Gal A-Fc protein in mouse serum was 471 min, approximately 43 times longer than that of the commercially available drug Replagal. These findings facilitate the development of an efficient production system for long-acting human α-Gal A-Fc fusion protein and provide valuable insights into the utilization of transgenic large animal mammary gland bioreactors for biopharmaceuticals.

Obesity is a well-established risk factor for male infertility. Recent studies have demonstrated that endoplasmic reticulum (ER) stress is a key contributor to spermatogenic disorder associated with obesity. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to mitigate ER stress, thereby alleviating insulin resistance. However, their specific role in obesity-induced reproductive disorders remains unclear. In this study, we used the transgenic fat-1 mice (TG mice) that are capable of endogenously converting Omega-6 polyunsaturated fatty acids (n-6 PUFAs) to n-3 PUFAs. The mice were divided into four groups according to their diet: a control group (WT + ND, n = 8), a wild type high-fat diet group (WT + HFD, n = 8), a transgenic control group (TG + ND, n = 8), and a transgenic high-fat diet group (TG + HFD, n = 8). After 18 weeks of feeding, the mice were anesthetized and euthanized to examine indicators related to obesity and reproductive function. High-fat diet (HFD) induced significant obesity in WT mice, and we observed significant alteration mitophagy in the reproductive function of WT mice (P < 0.001), primarily manifested as abnormal testicular morphology, decreased sperm quantity and motility (P < 0.01), and reduced testosterone levels (P < 0.01). TG mice exhibited a significant attenuation of these pathological changes (P < 0.05). Markers of ER stress and mitophagy were significantly reduced in the testes of TG mice (P < 0.01), accompanied by an increased expression of phosphorylated AMP-activated protein kinase (AMPK) (P < 0.01), compared to WT mice. Concurrently, TG mice exhibited significantly elevated levels of mitochondrial biogenesis markers and key enzymes involved in testosterone synthesis in the testes, compared to those in WT mice (P < 0.01). Furthermore, TG mice displayed notable resistance to testicular inflammation induced by HFD compared with WT mice (P < 0.01). Our findings suggest that HFD-induced obesity is associated with impaired testicular morphology and function in mice. n-3 PUFAs may ameliorate these impairments by activating AMPK to suppress ER stress, restore mitochondrial dysfunction, and alleviate inflammation, thereby improving testicular morphology and function.

Chia (Salvia hispanica L.), a nutritionally valuable crop, is the richest source of α-linolenic acid, a key omega-3 fatty acid. Despite its nutritional benefits, a stable genetic transformation method for chia is not available. This study presents a sonication-assisted Agrobacterium-mediated transformation protocol optimized for high-efficiency transformation of chia seedlings. Key parameters including bacterial cell density, acetosyringone concentration, sonication duration, vacuum infiltration, and infection time were optimized. Results demonstrated that an optical density at 600 nm (OD₆₀₀) of 0.5, acetosyringone concentration of 100 µM, 20 min of sonication, 10 min of vacuum infiltration, and 60 min of infection significantly enhanced transformation efficiency and GUS expression. This optimized protocol was validated through Polymerase Chain Reaction and β-glucuronidase (GUS) assay in transformed plants. Our findings establish a robust and reliable transformation protocol, paving the way for future genetic engineering efforts aimed at enhancing the nutritional and agronomic traits of chia.

Antimicrobial proteins and peptides are potential alternatives to antibiotics. Persulcatusin is an antimicrobial peptide found in the taiga tick Ixodes persulcatus. We constructed fusion genes that encode, from the N-terminus to the C-terminus, a signal sequence of rice α-amylase 3D, mouse calmodulin, a target sequence of TEV protease of tobacco etch virus, and persulcatusin with or without a His tag at the N-terminus of the mature fusion protein. These fusion genes were then introduced into rice. Western blot analysis detected persulcatusin fusion proteins in transgenic calli, suspension cells, and their culture medium. Antimicrobial activity against Staphylococcus aureus was detected in the protein extracts prepared from the transgenic callus but not from the non-transgenic wild-type callus, and TEV protease treatment to release persulcatusin from the fusion protein enhanced antimicrobial activity. The growth of the transgenic rice plants was unaffected. Our results indicate that functional persulcatusin can be produced in rice cells. This provides a basis for the mass production of persulcatusin for therapeutic use against bacterial infectious diseases in humans and livestock.