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Results for “Gibson assembly”

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PubMed2026

Generation of full-length wild-type and mutated futsch transgenes in Drosophila-efficient Gibson Assembly of ultra-large cDNAs.

Drosophila Futsch is a key microtubule-associated protein (fly homolog of MAP1B) that regulates microtubule organization, synaptic terminal growth, and neuronal development. Functional analysis of futsch has long been limited by the inability to clone and express a full-length futsch transgene, owing to its exceptional size (~16.5 kb) and extensive repetitive sequences. Here, I present an efficient and reproducible method for cloning both wild-type and mutated full-length Drosophila futsch cDNA (16,488 bp) using Gibson Assembly. These resulting cDNAs were used to generate UAS-futsch transgenes. When expressed in neurons, the wild‑type transgenic Futsch associated with microtubule and rescued the synaptic morphological defects observed in futschK68 mutants. This approach substantially reduces the time and complexity compared with traditional cloning techniques. Furthermore, I highlight common pitfalls encountered during the cloning process and provide practical solutions to enhance cloning efficiency. This protocol offers a broadly applicable and cost-effective framework for cloning otherwise intractable large cDNAs from Drosophila and other organisms.

Animals↗

PubMed2025

Rapid Generation of Reverse Genetics Systems for Coronavirus Research and High-Throughput Antiviral Screening Using Gibson DNA Assembly.

Coronaviruses (CoVs) pose a significant threat to human health, as demonstrated by the COVID-19 pandemic. The large size of the CoV genome (around 30 kb) represents a major obstacle to the development of reverse genetics systems, which are invaluable for basic research and antiviral drug screening. In this study, we established a rapid and convenient method for generating reverse genetic systems for various CoVs using a bacterial artificial chromosome (BAC) vector and Gibson DNA assembly. Using this system, we constructed infectious cDNA clones of coronaviruses from three genera: human coronavirus 229E (HCoV-229E) of the genus Alphacoronavirus, mouse hepatitis virus A59 (MHV-59) of Betacoronavirus, and porcine deltacoronavirus (PDCoV-Haiti) of Deltacoronavirus. Since beta coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome coronavirus (MERS-CoV) represent major human pathogens, we modified the infectious clone of the beta coronavirus MHV-A59 by replacing its NS5a gene with a fluorescent reporter gene to create a system suitable for high-throughput drug screening. Thus, this study provides a practical and cost-effective approach to developing reverse genetics platforms for CoV research and antiviral drug screening.

Reverse Genetics↗

PubMed2026

Prime assembly with linear DNA donors enables large genomic insertions.

Targeted insertion of large DNA fragments has promising applications for genome engineering and gene therapy1,2. Twin prime-editing guide RNAs have enabled relatively large insertions, but the efficiency remains low for insertions greater than 400 base pairs3-6. Here we describe a prime assembly (PA) approach for the insertion of large DNA donor fragments, of which the ends are designed to overlap with the flaps generated by twin prime editing (twinPE). We used PA to insert one or multiple overlapping DNA fragments, with total insertion sizes ranging from 0.1 kb to 11 kb. An inhibitor of non-homologous end joining enhanced both the efficiency and precision of insertions. PA relies on DNA templates that are easily produced, does not require co-delivery of exogenous DNA-dependent DNA polymerases and proceeds in non-cycling cells, suggesting independence from canonical homology-directed repair pathways. Our study demonstrates that PA can initiate Gibson-like assembly in cells to generate gene insertions without double-stranded DNA breaks, recombinases or homology-directed repair.

Animals↗

PubMed1979

Changes in collagen ultrastructure, macroscopic properties and chemical composition of chick embryo cartilage induced by administration of beta-D-xyloside.

Nine-day chick embryos were injected with a beta-xyloside and their sternal cartilage was examined 3 days and a week later. Sterna from 16-day embryos showed a reduction in size as compared to controls, with little or no change in the fraction of extracellular space, and a significant decrease in tensile strength. At the ultrastructural level, collagen fibrils in control sterna were dispersed evenly in the interstitial space, with few contacts between adjacent fibrils. In sterna from treated embryos, almost all collagen fibrils were aggregated into clumps and arrays throughout the interstitial space, with fibril-free areas in between. No abnormalities could be detected in the morphology of individual fibrils or in the ultrastructure of the chondrocytes. The changes in spatial distribution of collagen were fully evident 3 days after drug administration. The hydroxyproline/DNA ratio was the same in control and treated sterna, and no change was observed in the type of collagen. The uronic acid/DNA ratio was reduced by 14% 3 days after drug administration and by 40% after a week. The degree of sulfation of chondroitin sulfate was reduced from 80% in control sterna to 40% in treated sterna; almost all of this chondroitin sulfate was attached to peptide and the sedimentation pattern of the proteoglycan resembled that of normal cartilage proteoglycan. The function of chondroitin sulfate in embryonic cartilage is discussed in terms of our results and others. It is suggested that a major physiological role of the proteoglycan is to control the spatial distribution of collagen fibrils as they assemble to form a cross-linked gel.

Animals↗

PubMed2025

Single-cell-scale spatial transcriptome of the developing and adult mouse ovary.

Mammalian ovary development is essential for female fertility, involving the complex spatial patterning of diverse cell types to establish the finite reserve of ovarian follicles. While single-cell transcriptome analyses have provided important insights into the mechanisms driving specification and developmental trajectories of ovarian cells, they disrupt this crucial spatial context. To overcome this limitation, we used 10X Genomics Visium HD spatial transcriptomics to analyze the developing mouse ovary while maintaining its native cellular architecture. We captured all ovarian cell types at eight key fetal and postnatal timepoints, generating a near single cell resolution library of spatial gene expression across ovarian development. This comprehensive dataset allows analysis of dynamic transcriptional signatures associated with unique spatial patterning throughout development, including the establishment of cortex and medulla and assembly of ovarian follicles in each region. This dataset represents a fundamental resource for the investigation of regulatory mechanisms driving spatial patterning of the ovary and opens new avenues to explore the spatial determinants of female fertility and reproductive longevity.

Journal Article↗

PubMed1979

Emergency percutaneous transtracheal ventilation (PTV).

Ventilation through a percutaneous needle catheter inserted into the trachea has been advocated for resuscitation in acute upper airway obstruction. However, the customized, prefabricated equipment proposed by others for that use is not always available. We describe a device for percutaneous transtracheal ventilation (PTV) that can be rapidly assembled using available, inexpensive hospital supplies. With this device, moderate inflation pressures and flow rates of oxygen resulted in arterial PaO2 of greater than 300 torr and PaCO2 values of approximately 40 torr in dogs. PTV may be used as a life-saving measure in cases of acute upper airway obstruction when conventional airway management is unsuccessful.

Airway Obstruction↗

PubMed1979

Prediction of body temperatures during exercise in flying clothing.

A mathematical model has been used to describe experimental results for core and skin temperatures in subjects undergoing a rest/activity cycle in two aircrew clothing assemblies at two environmental temperatures (wet bulb, globe temperature (WBGT) indices of 25.9 and 28.9 degrees C). The model presented compares well with published data for subjects in standard aircrew equipment assemblies. Aircrew flying at a WBGT of 28.9 degrees C in chemical defence clothing may reach an unacceptable level of mean body temperature within 40 min and deep body temperature will rise at 1 degree C.hr-1. To prevent deterioration in flying performance during repeated sorties, an alteration in the work/rest activity pattern or the introduction of effective cabin or personal conditioning systems may be required,

Aerospace Medicine↗