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Genetic Manipulation of Human Marrow: Gene Transfer Using Retroviruses
Published in Adrian P. Gee, BONE MARROW PROCESSING and PURGING, 2020
Philip Hughes, R. Keith Humphries
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.
Protoplasts as Tools in the Study of Moss Development
Published in R. N. Chopra, Satish C. Bhatla, Bryophyte Development: Physiology and Biochemistry, 2019
Neil W. Ashton, Philip J. Boyd, David J. Cove, Celia D. Knight
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*
Published in Peter M. Gresshoff, Molecular Biology of Symbiotic Nitrogen Fixation, 2018
Reinhard Simon, Ursula B. Priefer
One disadvantage of pRK290 and pLAFR1 is their lack of insertional inactivation and their limited number of cloning sites. Therefore, derivatives have been constructed with additional selectable markers and single restriction sites without affecting their advantageous characteristics. Examples include pRK293, pRK310, and pRK311176 and the series of pVK vectors,177 which carry an additional Km resistance gene, which allows not only insertional inactivation, but also offers additional cloning sites. pVK101 (21.3 kb) and the cosmids pVKlOO and pVK102 (both 23 kb) have proved to be useful in a variety of cloning strategies. For example, complementation studies of Nif mutants of the broad host-range cowpea Rhizobium have been successfully carried out178 using pVKlOO as a cloning vehicle and mobilization from the E. coli mobilizing strain SM10. pVK102 has teen used to construct a cosmid bank of plasmid pRjaUSDA193, the symbiotic plasmid of R. fredii USDA193,179 and mutants of R. fredii USDA191, which induced pseudonodules, have been restored to the wild-type phenotype by introduction of a pVK102 cosmid clone of strain USDA201 via pRK2013 triparental matings.180
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.
A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies
Published in mAbs, 2018
Gargi Roy, Tom Martin, Arnita Barnes, Jihong Wang, Rod Brian Jimenez, Megan Rice, Lina Li, Hui Feng, Shu Zhang, Raghothama Chaerkady, Herren Wu, Marcello Marelli, Diane Hatton, Jie Zhu, Michael A. Bowen
Three plasmids were constructed for the co-expression of IgG and RMD: 1) A PCR product encoding the Pseudomonas RMD gene was cloned 3′ of a human CMV promoter in a proprietary vector containing a puromycin selection marker by Gibson assembly (pCLD-RMD). The IgG was expressed from a separate vector containing both HC and LC genes driven by the human CMV promoter, along with the GS selectable marker (pCLD-IgG). 2) A GS-IRES-RMD cassette was constructed using overlapping PCR to link GS to RMD with the EMCV-R IRES from pIRES (Takara-Clontech, Mountain View, CA) and then cloned into pCLD-IgG to generate pCLD-IgG GS-IRES-RMD.3) An IRES-RMD cassette was cloned downstream of the IgG LC of pCLD-IgG to generate pCLD-IgG LC-IRES-RMD.