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Nanoengineering Neural Cells for Regenerative Medicine
Published in Klaus D. Sattler, st Century Nanoscience – A Handbook, 2020
Christopher F. Adams, Stuart I. Jenkins
Currently, the most popular method of genetic manipulation is the use of viral vectors (used almost exclusively when engineering neural cells for combinatorial therapy), which offer high transduction efficiencies. However, viruses have several drawbacks including: safety issues, associated with toxicity to the target cells and oncogenicity of the transduced population due to insertional mutagenesis (especially relevant when using stem cells which have a capacity to self-renew, with risk of tumor formation); a limit to plasmid size in the most versatile vectors; and a complex, time-consuming method of application (Lentz, Gray and Samulski, 2011; Rogers and Rush, 2012). Additionally, genes introduced by retroviral transduction have been shown to undergo downregulation over time (Palmer et al., 1991). All of these disadvantages pose a considerable barrier to clinical and commercial adoption of stem cells transduced with viruses. Although considered safer than viruses, non-viral transfection is generally associated with low transfection efficiencies, especially in the case of lipofection and electroporation (Cesnulevicius et al., 2006; Tinsley, Faijerson and Eriksson, 2006) or high cell death, in the case of nucleofection (Cesnulevicius et al., 2006).
Deep Learning and Economic Prospects in Medical and Pharmaceutical Biotechnology
Published in Hajiya Mairo Inuwa, Ifeoma Maureen Ezeonu, Charles Oluwaseun Adetunji, Emmanuel Olufemi Ekundayo, Abubakar Gidado, Abdulrazak B. Ibrahim, Benjamin Ewa Ubi, Medical Biotechnology, Biopharmaceutics, Forensic Science and Bioinformatics, 2022
Charles Oluwaseun Adetunji, Kingsley Eghonghon Ukhurebor, Olugbemi Tope Olaniyan, Juliana Bunmi Adetunji, Gloria E. Okotie, Julius Kola Oloke
Guoyao and Fuller (2019) revealed that biotechnology-based breeding is the needed breakthrough to meet the increasing response for genetic traits and demand of quality pork protein. They grouped gene cloning by identical gene or genetic engineering to generate modified microorganisms or animals. It is known that cloning conserves the genetic trait or breed, while recombinant DNA technology for bioengineering uses multiple sources of genes. Genome editing utilizes insertion, deletion, and silencing to produce genetically modified products. Many tools of gene editing are presently deployed for biotechnological processes such as zinc finger nuclease, clustered regularly interspaced short palindromic repeats-associated nuclease-9 and transcription activator-like effector nuclease targeting cell, bacteriophages or plasmid via transfection like electroporation, lipid-based ligands, microinjection, and nucleofection. The authors revealed that the use of genetically modified pigs has been able to produce bacterial phytase, growth hormone, C. elegans fatty acid desaturases, fungal carbohydrases, uncoupling protein-1; lack of myostatin, CD163 or α-1,3-galactosyltransferase; and concluded that biotechnology provides opportunity to improve efficiency of swine generation plus develop a substitute to antibiotics in the nearest future. Fang et al. (2016) revealed that with the advancement in molecular biology tools, more development is being seen in DNA-based technologies to provide and promote quality and safe crop breeding products, increase agricultural output, and equally protecting the eco-environment. Hence, their role in modern day agro-science sector is becoming more important. The authors in their study described the utilization of biotechnological skills and bioengineering to improve agricultural yield such as molecular markers, genetic engineering, and genome editing.
Impact of exoD gene knockout on the polyhydroxybutyrate overaccumulating mutant Mt_a24
Published in International Journal of Biobased Plastics, 2021
Sandra Mittermair, Juliane Richter, Philipp Doppler, Kevin Trenzinger, Cecilia Nicoletti, Christian Forsich, Oliver Spadiut, Christoph Herwig, Maximilian Lackner
Before the transformation, the strain Mt_a24 was picked from the plate and cultivated in BG11 medium for 7 days to ensure the viability of the culture. The sample preparation for the electroporation was done according to [41]. The complete supernatant was removed after the last washing step and the pellet was re-suspended in 100 µL 1 mmol L−1 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.5). 70 µL cell suspension was mixed with 1–5 µg linear or plasmid DNA (diluted in ultrapure water) and pipetted into a cuvette (gap 0.1 mm, Laktan). The electroporation was carried out with the Amaxa General Nucleofection (Lonza) using the bacterial program 3. Immediately after the electroporation, the cells were re-suspended in 1 mL BG11 and transferred into 250 mL shaking flasks filled with 50 mL BG11 medium containing 10 mmol L−1 NaHCO3. The cells were incubated for 5 days at 30°C, 50 μmol photons m−2 s−1 and 60 rpm. The culture was harvested at 3,000 rpm for 20 min and re-suspended in 300 µL BG11. 50 µL and 150 µL of the culture were, respectively, plated on BG11 agar supplemented with 2.5 µg mL−1 Cm and 10 mmol L−1 NaHCO3. The culture was incubated at 30°C and 10 μmol photons m−2 s−1 for 2–4 weeks until single colonies occurred. Those single colonies were transferred onto new plates and tested via Multiplex-PCR using the OneTaq® Quick Load® DNA polymerase (NEB) and the primers 6–34 F, 10–15 F, and 10–16 R for the genomic integration of the knockout construct. The visualization was done on a 1% agarose gel. The primer sequences are summarized in Table 1.