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Familial Polyposis Coli: A Model for The Study of Promotion and Transformation
Published in Herman Autrup, Gary M. Williams, Experimental Colon Carcinogenesis, 2019
Inasmuch as the retroviruses can only transform cells after integration in the host genome, the higher transformation phenomenon may be due to the availability of more virus integration sites in the GS or FPC cells than present in the host genomes of “normal” or unaffected individuals. Also, a stable integration of viral genome may increase transcription of novel mRNA and proteins which may again influence the transformation efficiency of these cells.
Engineering the Plant Cell Factory for Artemisinin Production
Published in Tariq Aftab, M. Naeem, M. Masroor, A. Khan, Artemisia annua, 2017
Mauji Ram, Himanshu Misra, Ashish Bharillya, Dharam Chand Jain
To develop transgenic A. annua L. strains with a high content of artemisinin by modulating the expression of the previously mentioned genes, an efficient system of genetic transformation as well as regeneration of explants of A. annua L. should be in place. Vergauwe et al. (1996) developed an Agrobacterium tumefaciens -mediated transformation system for A. annua L. plants with high transformation rates (75% regenerants harboring the foreign gene). Artemisinin content in the leaves of regenerated plants was 0.17%, which is higher than that present in the leaves of normally cultured plants (0.11% DW). They further investigated the factors viz. , the age of explants, the A. tumefaciens strain, and plant genotype influencing the transformation efficiency (Vergauwe et al., 1998). Later, Han et al. (2005) established a high-efficiency genetic transformation and regeneration system for A. annua L. via A. tumefaciens .
ras Genes in Drosophila melanogaster
Published in Juan Carlos Lacal, Frank McCormick, The ras Superfamily of GTPases, 2017
In two different experiments human and Drosophila systems were intermingled. In one case a fusion between the N terminus of the activated c-Ha-ras oncogene and the C terminus of the Ras3 gene was constructed.5 The chimeric gene, driven by the promoter of the native c-Ha-ras protooncogene, was used to transfect Rat-1 cells. Transformed foci were obtained although the transformation efficiency was about 10% as compared with transfections in which intact activated c-Ha-ras DNA was used. A similar construct, but with the C terminus of Ras1 did not yield any transformed foci unless a viral promoter was added. In the latter case the transformation efficiency was 2% as compared with intact activated c-Ha-ras DNA. Since the C termini of the Drosophila Ras proteins differ greatly from the human ras sequence, it appears that the only contribution of the Drosophila sequences is to provide the required anchoring to the cell membrane, and to serve as a spacer between the membrane and the active N terminus.
Deciphering cross-species reactivity of LAMP-1 antibodies using deep mutational epitope mapping and AlphaFold
Published in mAbs, 2023
Tiphanie Pruvost, Magali Mathieu, Steven Dubois, Bernard Maillère, Emmanuelle Vigne, Hervé Nozach
Five human LAMP-1 libraries with single amino acid mutations were constructed using SOE-PCR and NNK codons. Library 1 corresponds to amino acids 29 to 99, library 2: amino acids 100 to 194, library 3: amino acids 195 to 226, library 4: amino acids 227 to 309 and library 5: amino acids 310 to 382. Following the mutagenesis, genes were constructed and amplified by SOE-PCR. Preparation of competent yeast cells EBY100 (ATCC® MYA-4941) and library transformation were performed according to Benatuil et al.51 Libraries were generated by gap repair cloning in yeast cells electroporated with 1 μg of digested vector and a molar ratio of 1.5:1 (library genes/digested vector). Transformation efficiency was determined by plating serial dilutions on selective agar plates. Each library contained at least 106 clones. Transformed cells were cultured for 2 d in SD-CAA medium (6.7 g/L yeast nitrogen base without casamino acids, 20 g/L glucose, 5 g/L casamino acids, 100 mM sodium phosphate, pH 6.0), at 30°C with shaking. After a passage to an OD600 of 0.25, cells were grown at 30°C until OD600 0.5–1.0 and re-suspended in 50 mL of SG-CAA for induction and incubated at 20°C.52
Sprifermin: a recombinant human fibroblast growth factor 18 for the treatment of knee osteoarthritis
Published in Expert Opinion on Investigational Drugs, 2021
Jia Li, Xiaoshuai Wang, Guangfeng Ruan, Zhaohua Zhu, Changhai Ding
However, restricted by the low expression and bioactivity level, it was difficult to produce FGF18 with full length [22]. Instead, utilizing specific structural properties of FGF18 could be a credible alternative considering the production and purification [23]. Thus, there was an emergent need for efficient expression systems of recombinant proteins to promote industrial production and pharmacological applications [24]. Among them, the Escherichia coli expression system emerged as the most productive and suitable one for large-scale manufacture, considering its high transformation efficiency, quick growth, but low cost [24]. Sprifermin (rhFGF18) was exactly a 19.83 kDa protein derived from E.coli, which consisted of 170 amino acid residues after C-terminal truncation of the full length protein [25].
Echinacea biotechnology: advances, commercialization and future considerations
Published in Pharmaceutical Biology, 2018
Jessica L. Parsons, Stewart I. Cameron, Cory S. Harris, Myron L. Smith
The discovery of R. rhizogenes-based hairy root transformation systems in higher plants provides other opportunities to engineer useful traits in Echinacea. Again, public acceptance of GMOs may limit the application of this useful technology. As an example, glufosinate-resistance and a fungal resistance chitinase gene were simultaneously transferred into E. purpurea using R. tumefaciens (Hanafy et al. 2010). Considering Echinacea plants in the field are particularly susceptible to weed competition and fungal pathogens, this study represents a useful demonstration model. Several factors are noted to influence Rhizabium-based transformation efficiency of Echinacea, and there is room for optimization. For example, the efficacy of the utilized bacterial strain is important; A4 strains were superior for transforming Echinacea leaf explants, whereas R1000 strains worked best with petioles (Wang et al. 2006). Overall, early development stages, such as cotyledon tissue, are more easily transformed and sonication is up to twice as effective for producing transformants compared to the traditional methods of wounding with a sterile needle to enhance R. rhizogenes-mediated gene transfer (Kumar et al. 2006). Addition of inducers to the medium during co-cultivation of agrobacterium with the plant tissue also improves efficacy. For example, indole-3-butyric acid (IBA) increases production of hairy roots in Echinacea by as much as 14 times (Romero et al. 2009). Other inducers of Rhizobium-associated gene transfer in plants (e.g., 6-benzylaminopurine, 2,4-dichlorophenoxyacetic acid) have been applied to Echinacea hairy root cultures to improve transformation but their effectiveness relative to no treatment has not been investigated empirically (Trypsteen et al. 1991; Wang et al. 2006).