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Gene Therapy for Acquired Tissue Damage
Published in Yashwant V. Pathak, Gene Delivery Systems, 2022
Rakesh Sharma, Robert Moffatt, Yuvraj Singh Negi, Shashi Prabha Singh
Prior infection or vaccination with related viruses may affect the safety and efficacy of the GTMP (e.g., adenoviruses, poxviruses [smallpox vaccine]); thus, the preexisting immunity to the vector itself should be determined prior to initiation of the therapy if a vector is chosen for which preexisting immunity can be assumed. These data might also determine the need for immune suppression. An immune response to the transgene product might eventually compromise the efficacy of the product and might have an impact on safety. Thus, evaluation of the immune response to the transgene product (i.e., determination of antibodies against the expressed protein) should also be part of the clinical development. In case repeated administration of the GTMP is foreseen, early considerations of the most appropriated vector (sero)type should be conducted, as well as the need for immune suppression of the patients. A comprehensive evaluation of the immune response to the vector and the transgene product has to be performed. This might include the evaluation of the cellular and humoral immunity to the vector as well as to the transgene product (e.g., titer and avidity of antibodies and information on whether the antibodies are neutralizing or not). The results should be documented in relation to the timing of the treatments, and correlation of the immunogenicity results with concurrent safety and efficacy should be provided.
Biomanufacture
Published in John M. Centanni, Michael J. Roy, Biotechnology Operations, 2016
John M. Centanni, Michael J. Roy
A transgenic plant or animal is a plant or animal that has been genetically altered using recombinant DNA techniques to create a genetically unique organism. The transgenic organism contains an exogenous gene or genes that have been intentionally inserted into their genome. Once inserted, the expression of the exogenous gene can express the protein of interest, often a glycoprotein, and, in some cases, secrete this protein with tissue fluid. In the case of a transgenic plant, the protein can then be extracted from biomass, that is, stems or leaves, or from seed. In case of transgenic animals, the protein is available from secretions, notably milk. Hence, the plant or animal functions as a bioreactor, producing appreciable amounts of the desired protein as BS. As one would anticipate, production of a transgenic organism capable of producing and secreting the perfect protein is highly technical and requires significant experience and skill. As with any biopharmaceutical, the protein product must be isolated and purified from other molecules and it must possess posttranslational modifications and structure that allow full biological function and should be without modifications that could make the molecule allergenic or nonfunctional. This process, using a transgenic goat secreting in milk, is provided in Figure 6.15.
Animal biotechnology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
To date, there are three basic methods of producing transgenic animals: DNA microinjection, retrovirus-mediated gene transfer, and embryonic stem (ES) cell—mediated gene transfer. Gene transfer by microinjection is the predominant method used to produce transgenic farm animals. Since the insertion of DNA results in a random process, transgenic animals are mated to ensure that their offspring acquire the desired transgene. However, the success rate of producing transgenic animals individually by these methods is very low and it may be more efficient to use cloning techniques to increase their numbers. For example, gene transfer studies revealed that only 0.6% of transgenic pigs were born with a desired gene after 7000 eggs were injected with a specific transgene. Although we benefit a lot from transgenic animals, still there are issues on how they are created, and these issues need to be resolved. The biotechnology industry, scientists, policy-makers, and the public cannot ignore thoughtful ethics, and ethical concerns must be addressed as technology grows, including the issue of the welfare of laboratory animals. Interestingly, the creation of transgenic animals has resulted in a shift in the use of laboratory animals from the use of higher-order species such as dogs to lower-order species such as mice and has decreased the number of animals used in such experimentation, especially in the development of disease models. This is certainly a good turn of events for transgenic technology holds great potential in many fields such as agriculture, medicine, and industry. In the next sections, we have listed different types of transgenic animals that are generated and used in various applications.
Increased removal of cadmium by Chlamydomonas reinhardtii modified with a synthetic gene for γ-glutamylcysteine synthetase
Published in International Journal of Phytoremediation, 2020
René Piña-Olavide, Luz M. T. Paz-Maldonado, M. Catalina Alfaro-De La Torre, Mariano J. García-Soto, Angélica E. Ramírez-Rodríguez, Sergio Rosales-Mendoza, Bernardo Bañuelos-Hernández, Ramón Fernando García De la-Cruz
The spectinomycin-resistant C. reinhardtii RPO2, successfully harvested in selective media, showed the transgenic state of this clone, as it has the gene aadA encoding for aminoglycoside adenyl transferase that confers such resistance. To confirm the presence of the transgene, a PCR analysis revealed the amplification of the targeted gene segment for gshA in DNA samples from one clone. In this analysis, the transplastomic clone RPO2 matched the positive control (+), which contained the amplification band (792 bp) of gene aadA, when compared to it and the two negative controls, one being the reaction without DNA (C–) and the other being the wild-type (WT) strain (Figure 2b). Furthermore, the amplified specific region of cDNA for the gene gshA, using sense Poli2Frt and antisense Poli2Rrt oligonucleotides by RT-PCR, related the sequence of 329 bp to the selected intergenic region for gshA in the characteristic location of this gene (Figure 2c). These results confirmed the presence of the construct pRPO2 in the transformed clone (RPO2) of C. reinhardtii.
Arsenic removal using Chlamydomonas reinhardtii modified with the gene acr3 and enhancement of its performance by decreasing phosphate in the growing media
Published in International Journal of Phytoremediation, 2019
Angélica E. Ramírez-Rodríguez, Bernardo Bañuelos-Hernández, Mariano J. García-Soto, Dania G. Govea-Alonso, Sergio Rosales-Mendoza, M. Catalina Alfaro de la Torre, Elizabeth Monreal-Escalante, Luz M. T. Paz-Maldonado
We successfully subcloned the optimized gene coding for acr3 into the pCAMBIA-1304 vector, with its expression mediated by the constitutive CaMV35S promoter, resulting in the vector called pARR1N. Selected positive clones by restriction profile analysis, confirmed the presence of this acr3 gene insert. We verified such construct by conventional sequencing, finding no other introduced variations in the coding sequence during the cloning procedure. Additionally, hygromycin-resistant C. reinhardtii clones, successfully harvested in selective media, indicated a transgenic state for these clones. Confirming the presence of the transgene, a PCR analysis revealed the amplification of the targeted acr3 gene segment in DNA samples from three clones, with no similar amplifications observed in the DNA sample from the wild-type strain (Figure 1a). To determine the transcription of the heterologous acr3 gene, a RT-PCR analysis performed to detect acr3 transcripts, revealed that the three clones were positive for genomic analysis yielding 145 bp amplicons, whereas such signal was absent in the case of cDNA from the wild-type strain. Finally, the amplification of β-tubulin transcripts led to homogenous amplicon bands (Figure 1b).
Development of an improved lentiviral based vector system for the stable expression of monoclonal antibody in CHO cells
Published in Preparative Biochemistry and Biotechnology, 2019
Omid Mohammadian, Masoumeh Rajabibazl, Es’hagh Pourmaleki, Hadi Bayat, Roshanak Ahani, Azam Rahimpour
The choice of expression vector and its regulatory elements can significantly affect transgene expression in mammalian cells. Lentiviralbased vectors provide attractive tools for in vivo and in vitro gene delivery to mammalian cells due to their high transduction efficiency, and stable-long term expression of transgene.[31]