Transgenic Research最新文献

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.

Soybean, a protein and oil rich legume is primarily used as livestock feed and to a lesser extent for human consumption due to undesirable flavour in the seeds caused by L-2 isozyme of lipoxygenase. Herein, soybean with reduced isozyme activity was developed through CRISPR/Cas9 targeted mutation in L-2 encoding Lox-2 gene. sgRNA designed from PLAT/LH2 domain in second exon of Lox-2 (Lox-2 E2) was validated by in vitro cleavage assay; inserted in CRISPR/Cas9 binary vector and used for genetic transformation of SL1074 cultivar hypocotyl segments. A total of 12 T₀ putative plants were identified through PCR. Amongst these, four revealed mutation at the target sgRNA site by CEL1 assay and substitution of a base A with G six bp upstream of PAM converting lysine to glutamic acid at 119 position. T₁ and T₂ seeds derived from mutant T0-37 plant showed upto 25.49% reduction in isozyme activity as compared to SL1074. The base substitution was confirmed in T₁ progeny; segregation analysis revealed homozygosity and heritability of mutation in T₂ plants. The interaction between structural models of SL1074, mutant domains and negatively charged substrates revealed strong binding affinity of the substrates with positively charged lysine in SL1074 domain due to formation of two hydrogen bonds. On the contrary, weak binding of the substrates with negatively charged glutamic acid in mutant domain and absence of hydrogen bond explained reduction of isozyme activity in T₂ seeds. The mutant soybean with reduced isozyme activity is an important source for introgressing the trait in plant breeding programs.

The advancement in the CRISPR/Cas system has significantly streamlined genome editing in plants, rendering it simple, reliable, and efficient. However, the development of transgene-free crops is a challenging task for vegetatively propagated plants like banana. In the present study, we established banana protoplasts-based versatile and efficient platform for genome editing to overcome this limitation. Herein, a protocol has been optimized for protoplast isolation by considering leaf and embryogenic cell suspension (ECS) of banana cultivar Grand Naine. Freshly prepared ECS was identified as the best source for protoplast isolation. The protoplast viability and competency were checked by transfection with plasmid and RNP complex. Polyethylene glycol (PEG)-mediated protoplast transfection using pCAMBIA1302 and pJL50TRBO vectors showed GFP expression with 30 and 70% efficiency, respectively, eventually proving the protocol's efficacy. Further, gRNAs targeting banana β-carotene hydroxylase gene are validated by in-vitro cleavage test and subsequently used for RNP complex formation with varied ratios (1:1, 1:2, 1:5, and 1:10) of SpCas9 to gRNA1. Among these, a 1:2 molar ratio proved best to generate indel frequency with 7%. Sequencing analysis of the target amplicon revealed mutations upstream of the PAM region, specifically with gRNA1, among the three in-vitro validated gRNAs. This study evaluated the effectiveness of gRNAs in-vitro and in-vivo, yielding inconsistent results that highlight the need for comprehensive in-vivo validation of their functionality. Conclusively, the optimized protocol for banana transfection has the potential to be harnessed for the generation of transgene-free genetically improved banana.

The Agave genus is recognized for its diversity, economic importance, and adaptability to arid and semi-desert climates. Among its species, Agave sisalana stands out for the production of sisal, a resilient natural fiber with various industrial applications and potential to be used as a raw material in biofuel production, due to the accumulation of fermentable sugars in its biomass. Although this species presents significant agronomic potential, challenges in conventional breeding hinder its cultivation. A viable alternative for genetic improvement is the development of transgenic plants that incorporate desirable agronomic traits through the controlled insertion of genes of interest into their genome. The objective of study was to develop a protocol for obtaining transgenic plants of Agave sisalana 'RLV19,' a species widely cultivated in the semi-arid region of Bahia-BA, Brazil, using the cp4-epsps gene aimed at constitutive gene expression. Two transgenic plants of Agave sisalana 'RLV19' were regenerated via organogenesis. PCR analyses and CP4-EPSP protein expression by RT-PCR confirmed the presence and expression of the transgene in these plants. This is the first report of A. sisalana transgenic plants expressing the cp4-epsps gene, and to the best of our knowledge, there have been no prior reports on protocols for the production of transgenic plants of this species.

Horticultural crops, with their cost-effectiveness, rich mineral and vitamin content, and high yield potential, have become indispensable worldwide for ensuring food and nutritional security. With the world's population on the rise and climate change becoming more prominent, it is crucial to focus on creating resilient, high-yielding crop varieties that can withstand the changing climate. Genetic improvement of different horticultural crops using conventional tools is both time-consuming and labourious. However, the breeding period can be cut short by adopting modern breeding techniques, including CRISPR/Cas-mediated genome editing. In the present review, we discuss the progress made so far through genome editing to improve several horticultural crops for various traits like stress resistance, morphology, nutritional attributes, quality, shelf life, male sterility, architecture and economic yield. We have also discussed the emerging CRISPR technologies like base editing, epigenome editing, CRE editing, transposon-based editing, prime editing etc., along with their pros and cons and the future prospects. The ethical considerations for commercialization and current regulatory frameworks for gene-edited products have also been discussed.

Potassium is a vital element in sweetpotato that plays important roles during its growth and development. In this study, potassium transporter IbHAK5, which is homologous to Arabidopsis HAK5, was cloned and overexpressed in sweetpotato. IbHAK5 encoded a protein of 739 amino acids and localized in the plasma membrane. Two IbHAK5-overexpressing transgenic lines with the highest expression level of IbHAK5 were screened for K⁺-deficiency stress tolerant assay. Compared with wild type sweetpotato plants, transgenic plants grew well with higher chlorophyll content, and maintain great higher K⁺ contents via decreasing more K⁺ effluxes under low potassium ion (- K⁺) stress condition. Additionally, IbHAK5 can help plants improve root morphology and increase endogenous hormone IAA content under both normal condition and - K⁺ stress, which may result in the increased root K⁺ absorption ability. The results indicated that IbHAK5 play an important role in sweetpotato response to - K⁺ stress, as well as support molecular-assisted breeding with the IbHAK5 gene.