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Embryo Cell-Free DNA in the Culture Medium and Its Potential for Non-Invasive Aneuploidy Testing
Published in Carlos Simón, Carmen Rubio, Handbook of Genetic Diagnostic Technologies in Reproductive Medicine, 2022
Carmen Rubio, Luis Navarro-Sánchez, Carmen M. García-Pascual
The greatest methodological challenge of niPGT-A is avoiding contamination with DNA that does not come from the embryo. This exogenous DNA can come from the cumulus cells (maternal cells), or from external sources (e.g., technicians, contaminated media, working surfaces, or material) [16,17,34]. Strategies to minimize the carryover of cumulus cells into culture drops include gentle denudation after IVF or ICSI and extra serial washes. Individual handling of embryos, sterile working conditions, and the use of dedicated materials throughout the procedure also reduce the risk of contamination.
Gene Therapy for Chronic Inflammatory Diseases of the Lungs
Published in Kenneth L. Brigham, Gene Therapy for Diseases of the Lung, 2020
Most of the research in gene therapy for chronic inflammatory lung disease has focused on alpha1 antitrypsin deficiency and cystic fibrosis. Significant advances have been made in the field of gene therapy for lung diseases. Current work has shown that exogenous DNA can be transferred to the lungs of animals and humans and function in a physiologically appropriate manner. Yet, much more research needs to be done. Most importantly, improvement in gene delivery vectors will be necessary for this new therapeutic intervention to realize its full potential.
Preimplantation Genetic Testing for Structural Rearrangements
Published in Darren K. Griffin, Gary L. Harton, Preimplantation Genetic Testing, 2020
Short-tandem repeat (STR) typing involves the application of a PCR-based protocol using multiplexed STR markers located on both sides of the breakpoint [68]. An extensive workup is required specific to the translocation in question. A total of 29 patients were analyzed by this method and the proportion of alternate segregation for RecT and RobT was 33% and 77%, respectively [68]. The fetal heartbeat rate was 46% for RecT and 40% for RobT carriers. This technique might also provide an additional control for contamination of exogenous DNA, detection of uniparental disomy (UPD), and the possibility of adding extra markers for aneuploidy for other chromosomes [69]. In another study, a total 241 embryos were analyzed from 27 couples using a similar multiplex STR system. In this cohort, the frequency of alternate segregations was 38.5% and 66.1% for RecT and RobT carriers, respectively. A total of 90 embryos were also tested for aneuploidy for 9 other chromosomes (13, 14, 15, 16, 18, 21, 22, X, Y) in addition to the rearrangement, which showed that 63.1% of them were aneuploid. A relatively high rate of implantation (59.6%) was achieved possibly due to additional aneuploidy testing on the normal/balanced embryos [69]. Although these studies demonstrate the reliability of the approach, there are drawbacks in the time and cost of that workup.
How necessary are animal models for modern drug discovery?
Published in Expert Opinion on Drug Discovery, 2021
Gene therapy tools for adding or inserting an exogenous DNA copy into the target cell nucleus or genome may lead to side effects, as insertional mutations or latent (post-translation) expression of proteins. The programmable nucleases use a ‘cut-and-paste’ approach to remove the defect and install the correct gene. An RNA-guided genome editing tool known as clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR-Cas9) provides numerous advantages over the conventional gene therapy and demonstrates therapeutic promises [22]. CRISPR-Cas9 can be used in several ways for therapeutic purposes. It can correct the mutations and rescue the disease phenotypes. It can also engineer pathogen genomes for therapeutic purposes or induce protective or therapeutic mutations in host tissues. It has demonstrated promise in cancer gene therapy by deactivating oncogenic virus and inducing oncosuppressor expressions. The fast-growing use of CRISPR-Cas9 technology is appearing as an effective tool for the characterization and treatment of various human diseases. With time, this technology may revolutionize gene therapy and become a versatile option for gene therapy.
Delivering CRISPR: a review of the challenges and approaches
Published in Drug Delivery, 2018
Christopher A. Lino, Jason C. Harper, James P. Carney, Jerilyn A. Timlin
The use of site-specific nucleases and NHEJ or HDR generally results in one of four gene editing products. Shown in Figure 2, these include gene knockout, deletion, correction, or addition. The error-prone character of NHEJ can be exploited to introduce indels and frameshifts into the coding regions of a gene. This knocks the gene out (Figure 2(A)) via nonsense-mediated decay of the mRNA transcript. In gene deletion (Figure 2(B)), paired nucleases excise regions of the coding gene, resulting in premature truncation and knockout of the protein in a manner more generally efficient than introducing frameshifts. Both gene correction (Figure 2(C)) and gene addition (Figure 2(D)) require an exogenous DNA template that can be introduced as either single-stranded (Radecke et al., 2010; Chen et al., 2011; Soldner et al., 2011) or double-stranded DNA (Rouet et al., 1994). The DNA template contains homologous sequence arms that flank the region containing the desired mutation or gene cassette.
Advanced physical techniques for gene delivery based on membrane perforation
Published in Drug Delivery, 2018
Xiaofan Du, Jing Wang, Quan Zhou, Luwei Zhang, Sijia Wang, Zhenxi Zhang, Cuiping Yao
The applications of magnetoporation in vitro and in vivo have been reported and shown better efficiency, such as cardiac tissue (Li et al., 2008), skeletal muscle (Zhou et al., 2007; Pereyra et al., 2016), liver tumors (Almstäetter et al., 2015), mouse myoblast (Akiyama et al., 2010), and mouse brain (Hashimoto & Hisano, 2011; Sapet et al., 2012; Soto-Sanchez et al., 2015). Moreover, magnetoporation is often used to transfect the difficult-to-transfect cells. Pereyra et al. (2016) recombined the adenoviral vectors with iron oxide nanoparticles into magneto-adenovectors to transfect the C2C12 myotubes in vitro and mouse skeletal muscle in vivo. Their results demonstrated that the magneto-adenovectors could improve the transfection rate of myotubes and enhance the transfection efficiency of muscle cells. Central nervous system is difficult to transfect under the effect of static magnetic field (Pickard et al., 2011). Therefore, Adams et al. (2013) employed the oscillating magnetic fields to transfect the neural stem cell, and over two-fold transfection efficiency was acquired. Cui and his teammates devoted to research transfection of animal cells through magnetofection (Wang et al., 2013; Y. Wang et al., 2014; Zhao et al., 2014; Chen et al., 2015). Recently, they reported a successful study on magnetoporation in plant transformation. Firstly, they introduced the exogenous DNA into the pollen under the effect of magnetic field. Then, the transfected seeds were successfully generated by pollination. Further, the exogenous DNA was successfully transferred into plant cell and expressed in the offspring (Zhao et al., 2017).