Protoplasts as Tools in the Study of Moss Development
R. N. Chopra, Satish C. Bhatla in Bryophyte Development: Physiology and Biochemistry, 2019
We are currently engaged in confirming P. patens transformation by the Southern hybridization technique.34,39 There is one peculiarity of the selection of kanamycin-resistant transformants in P. patens which has so far prevented us from obtaining sufficient tissue from a number of regenerants for DNA isolation. After primary selection on kanamycincontaining medium, strongly resistant colonies are unable to regenerate normally after subculture. However, transfer of undisturbed regenerants to fresh kanamycin does not inhibit growth.32 Because of this and because many inocula are also unable to survive transfer to minimal medium it is unlikely that the transformants are genetically unstable. Instead it seems that kanamycin or the presence of a kanamycin resistance gene may interfere with regeneration of chloronemal cells. Although this may indicate that selection for kanamycin resistance is unsuitable for P. patens, there may be ways of overcoming this problem by allowing caulonemal cells to develop before application of the antibiotic. Alternative selectable marker genes are also under consideration,40 as are alternative transformation procedures.
Vector Technology of Relevance to Nitrogen Fixation Research*
Peter M. Gresshoff in Molecular Biology of Symbiotic Nitrogen Fixation, 2018
The mutation caused by the insertion of a drug-resistance transposon is accompanied by the acquisition of a selectable marker. Thus, after transposon mutagenesis, a cell carrying an insertional mutation is positively selectable; nonmutated cells will not survive the selection by drug resistance for the presence of the transposon. This is in striking contrast to classic mutagenesis procedures. For example, in practice it is not possible to apply chemical or physical mutagens at doses which would result in one mutation per surviving cell; low-level mutagenesis results in a high proportion of unmutated cells. On the other hand, heavy mutagenic treatment results in the high frequency of multiple mutational events, an undesirable point in genetic analysis.
Genetic Manipulation of Human Marrow: Gene Transfer Using Retroviruses
Adrian P. Gee in BONE MARROW PROCESSING and PURGING, 2020
Retroviral vectors need not contain selectable markers. If the retroviral construct contains a gene that codes for a secreted product for which there is an assay, for example, a growth factor gene and a cell proliferation assay for that growth factor, then one can screen for clones of packaging cells that are best at converting 3T3 cells to growth factor production.10,11 Similarly, if the retroviral gene codes for a cell surface marker for which an antibody exits, then one can screen for clones that produce targets with the most cell surface protein.12 Both these methods are much more arduous than using a selectable marker, but have the advantage of looking directly at the gene of interest.
Broad reactivity and enhanced potency of recombinant anti-EGFR × anti-CD3 bispecific antibody-armed activated T cells against solid tumours
Published in Annals of Medicine, 2022
Manley T. F. Huang, Vikram Sharma, Andrew Mendelsohn, Qisheng Wei, Jinjing Li, Bo Yu, James W. Larrick, Lawrence G. Lum
The variable light and heavy gene sequences from the DrugBank amino acid entry for Erbitux were back-translated using the most common homologous mouse sequences in the IMGT database. The variable light and heavy chain genes for humanized muromonab-CD3 were designed based on amino acid sequences in US patent 5,885,573. The OKT3 scFv-Erbitux-heavy chain fusion gene was assembled in the order: variable light chain – (G4S)6 linker-variable heavy chain – (G4S)5 (with a change in repeat 3 to introduce a G to T substitution) linker – Erbitux heavy chain variable sequence - human IgG1 Fc. Coding regions for the Erbitux light and OKT3scFv-Erbitux fusion genes were then subcloned into the proprietary expression vector SwiMR (US9910038B2) which contains selectable markers for puromycin or neomycin. The amino acid sequences for the Erbitux light chain and humanized OKT3-Erbitux heavy chain fusion chain expression cassettes are shown in Figure 4.
Green synthesis of silver nanoparticles using transgenic Nicotiana tabacum callus culture expressing silicatein gene from marine sponge Latrunculia oparinae
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Yuri N. Shkryl, Galina N. Veremeichik, Dmitriy G. Kamenev, Tatiana Y. Gorpenchenko, Yulia A. Yugay, Dmitriy V. Mashtalyar, Aleksander V. Nepomnyaschiy, Tatiana V. Avramenko, Aleksandr A. Karabtsov, Vladimir V. Ivanov, Victor P. Bulgakov, Sergey V. Gnedenkov, Yury N. Kulchin, Yury N. Zhuravlev
Agrobacterium tumefaciens carrying pPZP-RCS2-nptII, pPZP-RCS2-nptII/LoSilA1, pPZP-RCS2-nptII/LoSilA1-EGFP and pPZP-RCS2-nptII/EGFP plasmids were used to inoculate leaf discs of sterile, clonally cultivated plantlets of N. tabacum L. (cv Xanthi) according to the previously described conditions [42]. Leaf discs were co-cultivated with A. tumefaciens for 48 h and transferred to the WB/A agarized medium supplemented with 0.5 mg/L 6-benzylaminopurine and 2.0 mg/L α-naphthaleneacetic acid [43] containing 500 mg/L cefotaxime and 100 mg/L kanamycin. Transgenic callus cultures expressing the selectable marker (nptII) gene alone or together with the LoSilA1 gene, LoSilA1-EGFP fusion gene or EGFP gene, namely Nt-cV, Nt-cS, Nt-cSG and Nt-cG, were obtained after four months of selection with kanamycin. Plant regeneration was achieved by placing the 4–5 week-old primary calli on hormone-free W medium containing 100 mg/L kanamycin under a 16/8 h light/dark cycle. Thus, transgenic plantlets expressing the selectable marker (nptII) gene alone or together with the LoSilA1 gene, LoSilA1-EGFP fusion gene or EGFP gene, namely Nt-pV, Nt-pS, Nt-pSG and Nt-pG, were obtained. The control non-transformed callus culture and plants were established from the same plantlets and cultivated under the same conditions as the transformed ones.
Current advances in the algae-made biopharmaceuticals field
Published in Expert Opinion on Biological Therapy, 2020
Sergio Rosales-Mendoza, Karla I. Solís-Andrade, Verónica A. Márquez-Escobar, Omar González-Ortega, Bernardo Bañuelos-Hernandez
It has been proposed that poor transgene expression is derived from severe transcriptional silencing irrespective of the genomic position [52]. Gene silencing is a process where DNA methylation has important implications. It has been reported that a DNA methyltransferase (MET I) is involved in gene silencing in algae and the expression of the GFP(VENUS) protein in C. reinhardtii MET I null-mutant strains was higher than in the Chlamydomonas wild type [53]. An approach to overcome the epigenetic mechanisms that cause poor gene expression involves the development of mutant strains obtained by UV mutagenesis, which led to the generation of the UVM11 strain with enhanced capacity for transgene expression; allowing yields that were about 10-fold higher than the typical yields attained under conventional nuclear expression approaches [54]. This approach has been tested in combination with codon optimization. Barahimipour et al. [49] assessed the expression capacity of P24 and nptII gene variants that encode the identical amino acid sequence but differ in codon usage; using the mutant expression strain UVM11 and the wild type C. reinhardtii strain. The report showed that a codon-optimized P24 gene variant, introduced into the UMV11 strain, led to recombinant protein accumulation levels up to 0.25% of the total cellular protein. However, no wild-type strain was used to attribute the increase to the UMV11 strain. Moreover, in combination with an expression strain, a resynthesized nptII gene becomes a highly efficient selectable marker gene that allows selection of transgenic algal clones at high frequency [49].