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Adeno-Associated Virus-Based Delivery Systems
Published in Kenneth L. Brigham, Gene Therapy for Diseases of the Lung, 2020
The preintegration site has been cloned and inserted into an Epstein-Barr virus-based shuttle vector (105) for delivery to the cell. AAV infection led to integration in this episomal shuttle vector when it contained certain critical elements mapping to a 510-bp sequence upstream from the actual integration site. Three sequences present both in the preintegration site and in the AAV have been identified—a (GCTC)3 that is a binding site for Rep 68 and 78; a terminal resolution site that is nicked by rep; and a sequence homologous with a yeast recombination enhancer called M26. Others also have data suggesting that a 4-nucleotide repeat in the host genome is associated with integration in the presence of reps 78 and 68 (105,107).
Structural Determination of the Polycystin-2 Channel by Electron Cryo-Microscopy
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
To generate the recombinant baculovirus containing a polycystin-2 expression cassette, DH10Bac E. coli strain is transformed with the donor pFastbac-PKD2 plasmid. The DH10Bac E. coli strain contains a baculovirus shuttle vector (bacmid with a Tn7 attachment site) and a helper plasmid expressing transposase. Upon transformation, the target gene from the donor vector transposes to the low copy baculovirus shuttle vector at a mini-attTn7 transposon recombination site to generate high molecular weight bacmids (>135 kb in size, 1–5 copies per cell). Positive clones are then selected on an LB plate with multiple antibiotics under blue-white selection. As a result, success in bacmid production can be accessed by a small-scale growth from white colonies on selection plates and PCR validation using M13 forward and reverse primers. Recombinant baculovirus is produced in Spodoptera frugiperda (Sf9) cells and harvested in the cell culture medium.
Mycobacterium tuberculosis
Published in Lloyd N. Friedman, Martin Dedicoat, Peter D. O. Davies, Clinical Tuberculosis, 2020
In contrast to Neurospora, which was a diploid organism that underwent meiosis, transfer of DNA directly into Streptococcus was called transformation. The question as to whether bacteria had genes was answered convincingly when Salvador Luria and Max Delbruck used phages (bacterial viruses) as selecting agents, providing evidence that Charles Darwin's survival of the fittest premise existed even for bacteria.14 Gene transfer studies exploded when Joshua Lederberg and Edward Tatum demonstrated in 1946 that Escherichia coli could transfer genes from cell to cell (conjugation) using double auxotrophic mutants.15E. coli would become the model organism for molecular genetics for the next 30 years. The lingering debate as to whether DNA, not protein, was the genetic material was convincingly proved by Hershey and Chase's experiment with phages, bacteria, and radiolabeled DNA or protein.16 Notably, Norton Zinder, while trying to repeat the Lederberg experiment for Salmonella, discovered phages could transfer genes to other bacterial cells—a process he named transduction.17 Thus transformation, conjugation, and transduction became the key tools for the successful fulfillment of the third condition of Molecular Koch's postulate. The slow growth and virulence precluded successful gene transfer in M. tuberculosis until 1987.18 Using the first chimeric mycobacteriophage−E. coli shuttle vector, termed a shuttle phasmid—it became possible to transfer genes in M. tuberculosis (Figure 3.1). Nearly 100 years later, plasmid transformation, efficient transposon mutagenesis systems, specialized transduction, and reporter mycobacteriophages have enabled the acquisition of many phenotypes of M. tuberculosis, opening the doors for new therapeutic approaches (see reviews19–21).
Recombinant bacteriophage T4 Rnl1 impacts Streptococcus mutans biofilm formation
Published in Journal of Oral Microbiology, 2021
Juxiu Chen, Zhanyi Chen, Keyong Yuan, Zhengwei Huang, Mengying Mao
The oligonucleotides used in this study are presented in Table 2. All primers designed using MacVector 7.0 software were purchased commercially (Sangon Biotech, Shanghai, China). S. mutans UA159, referred to as the wild type (WT) strain, was used as a recipient for mutant construction. The shuttle vector pDL278 was used to express the t4rnl1 gene under the control of the S. mutans endogenous promoter region of the vicR gene as previous study described [7]. The t4rnl1 and vicR promoter sequences were obtained by oligonucleotide synthesis (Sangon Biotech, Shanghai, China). The 1,495-bp amplicons were cloned into the vector pDL278 at Smal and SalI restriction sites generating recombinant plasmid pDL278_t4rnl1. The pDL278 blank and the pDL278_t4rnl1 plasmids were, respectively, transformed into WT strains. The resulting t4rnl1 expressed WT strain was named WT_T, while the one carrying pDL278 blank vector was named WT_B. Mutant strains were isolated on BHI-spec agar plates (spectinomycin, 1 mg/mL) and were further verified by colony PCR.
Heterologous prime-boost immunization co-targeting dual antigens inhibit tumor growth and relapse
Published in OncoImmunology, 2020
Qianqian Guo, Lizheng Wang, Ping Xu, Fei Geng, Jie Guo, Ling Dong, Xin Bao, Yi Zhou, Mengfan Feng, Jiaxin Wu, Hui Wu, Bin Yu, Haihong Zhang, Xianghui Yu, Wei Kong
An sPD1-MUC1-survivin fusion gene, used in our previous study,20 was inserted into the shuttle vector pSC11M1, and termed as pSC11M1-MS. The sPD1 protein was utilized to improve the cross-presentation of antigens. Homologous recombination was performed in BHK-21tk-ts13 cells for the production of recombinant MVA, according to the standard procedures. In brief, the BHK-21 tk-ts13 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco, Thermofisher), infected with WT-MVA at an MOI of 0.05, and transfected with pSC11M1-MS 2 h post-infection. The rMVA was selected by blue-white selection; 5-bromodeoxyuridine was used for thymidine kinase (TK) selection and recombinant MVA amplification. Zonal sucrose gradient centrifugation was used for purification of rMVA. The rMVA titer was determined via immunostaining with an anti-vaccinia antibody (Fitzgerald) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (Jackson ImmunoResearch).
Bioengineering commensal bacteria-derived outer membrane vesicles for delivery of biologics to the gastrointestinal and respiratory tract
Published in Journal of Extracellular Vesicles, 2019
Ana L. Carvalho, Sonia Fonseca, Ariadna Miquel-Clopés, Kathryn Cross, Khoon-S. Kok, Udo Wegmann, Katherine Gil-Cardoso, Eleanor G. Bentley, Sanaria H.M. Al Katy, Janine L. Coombes, Anja Kipar, Regis Stentz, James P. Stewart, Simon R. Carding
A 1018 bp chromosomal DNA fragment upstream from BT_3852 and including the first 18 nucleotides of its 5ʹ-end region was amplified by PCR using the primer pair f-5ʹompA_SpHI, r-5ʹompA_SalI. This product was then cloned into the SpHI/SalI sites of the E. coli-Bacteroides suicide shuttle vector pGH014 [20]. A 761 bp chromosomal DNA fragment downstream from BT_3852, including the last 46 nucleotides of the 3ʹ-end region, was amplified by PCR using the primer pair f-3ʹompA_BamHI, r-3ʹompA_SacI and was cloned into the BamHI/SacI sites of the pGH014-based plasmid. The resulting plasmid containing the ΔBT_3852::tetQ construct, was mobilized from E. coli strain GC10 into Bt by triparental filter mating [19], using E. coli HB101(pRK2013) as the helper strain. Transconjugants were selected on BHI-haemin agar containing gentamicin (200 mg/L) and tetracycline (1 mg/L). Determination of susceptibility to either tetracycline or erythromycin was done to identify recombinants that were tetracycline resistant and erythromycin susceptible after re-streaking transconjuguant bacteria on LB-agar containing tetracycline or both antibiotics. PCR analysis and sequencing were used to confirm allelic exchange. A transconjugant, GH290, containing the ΔBT_3852::tetQ construct inserted into the Bt chromosome was selected for further studies.