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Cell Line Development
Published in Wei-Shou Hu, Cell Culture Bioprocess Engineering, 2020
A high degree of gene amplification through the application of a high concentration of selective pressure often gives rise to phenotypically and genotypically unstable producing cells. It is advantageous to accomplish the same high level of transcription using a lower degree of amplification. A number of methods aim to attain high expression levels with a low multiplicity of the GOI. DHFR-selectable markers are used in conjunction with an impaired neomycin resistance gene. After plasmid transfection and antibiotic G418 selection, only cells to whose genome the vector has integrated in a transcriptionally active region and that give a high level of neomycin resistant transcripts can survive. The method thus allows for the selection of cells that the GOI has integrated into in a very transcriptionally active region known as a “hot spot” region. The amplification is then performed at a lower level of the DHFR inhibitor MTX. Since the locus of integration is transcriptionally active, the high-expressing clones isolated have only a few copies of the GOI.
Cytological and Histological Analysis by Image Processing
Published in Corain Livio, Arboretti Rosa, Bonnini Stefano, Ranking of Multivariate Populations, 2017
Corain Livio, Arboretti Rosa, Bonnini Stefano
Primary cell cultures were derived from foetal bovine cerebellum. Male bovine foetuses at 4 months were obtained at nearby commercial abattoirs on accidental slaughtering of pregnant cows. Animals were treated according to the European Community Council Directive (86/609/EEC) concerning animal welfare during the commercial slaughtering process and were constantly monitored under mandatory official veterinary medical care. Age was determined by crown-rump length based on commonly accepted reference tables (McGeady et al., 2006). The use of bovine foetal cells is a reliable tool for studying the molecular mechanisms of Ca2+ regulation and thus follows the joint declaration of the American, European and Japanese Societies for Neuroscience that endorses replacement of laboratory animals whenever a valuable scientific alternative model is available. Primary cell culture was performed following an established laboratory protocol (Peruffo et al., 2004). To obtain a stable cell line, cells were immortalized following the protocol by Takenouchi et al. (2007). At day 2 in culture, cells were transfected with pSV3neo plasmid (LGC Promochem, Teddington, UK) by using the cationic lipid Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Subsequently, a selection medium was added and resistant cells were selected with the antibiotic G418 (400 μkg/ml; Gibco, Life Techologies BRL).
Agricultural biotechnology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
In order to select only cells that have actually incorporated the new genes, the genes coding for the desired trait are fused to a gene that allows selection of transformed cells, so-called marker genes. The expression of the marker gene enables the transgenic cells to grow in the presence of a selective agent, usually an antibiotic or a herbicide, while cells without the marker gene die. One of the most commonly used markers is the bacterial aminoglycoside-3′ phosphotransferase gene (APH(3′)II), also referred to as neomycin phosphotransferase II (NPTII). This gene codes for an enzyme that inactivates the antibiotics kanamycin, neomycin, and G418 through phosphorylation. In addition to NPTII, a number of other antibiotic resistance genes have been used as selective markers, such as hygromycin phosphotransferase gene conferring resistance to hygromycin. Another group of selective markers is herbicide tolerance genes. Herbicide tolerance has been obtained through the incorporation and expression of a gene that either detoxifies the herbicide in a similar manner as the antibiotic resistance gene products or expresses a product that acts like the herbicide target but is not affected by the herbicide. Herbicide tolerance may not only serve as a trait useful for selection in the development of transgenic plants but also has some commercial interest. Transformation of plant protoplasts, cells, and tissues are usually only useful if they can be regenerated into whole plants. The rate of regeneration varies greatly not only among different species but also between cultivars of the same species. Besides the ability to introduce a gene into the genome of a plant species, regeneration of intact, fertile plants out of transformed cells or tissues is the most limiting step in developing transgenic plants (Figure 6.9).
Investigation of Dependency of Boric Acid and Lithium Metaborate Induced Yeast Toxicity on the Expressions of Antioxidative and Apoptotic Genes
Published in Egyptian Journal of Basic and Applied Sciences, 2023
Berna Kavakcıoğlu Yardımcı, Zehra Mollaoğlu, Fatih Altıntaş, Ayşenur Güler
For the growth of all the strains, YPD broth consisting of 10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose (pH: 5.6) was used. Geneticin antibiotic (G418), 200 mg/L dH2O, was also added to the growth medium of mutant strains. The inoculum was carried out in 250 mL Erlenmeyer flasks containing 95 mL broth plus 5 mL spore suspension whose optical density at 660 nm was 0.1. The yeast cells were grown until their early-exponential phase with 180 rpm agitation at 30°C. After the cells reached the desired density, the cells were treated with the agents as previously described [20]. Briefly, BA and LBM were added to the flasks at the final concentrations of 0.25–1% and 0.05–0.25%, respectively, and the cells were additionally incubated 12 and/or 36 h under the same conditions. At the end of the final incubation period, the cells were harvested with a centrifuge for 5 min at 3,000 g at 4°C and were kept at −80°C until further biochemical analysis.