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Should Genome Editing Replace Embryo Selection Following PGT?
Published in Carlos Simón, Carmen Rubio, Handbook of Genetic Diagnostic Technologies in Reproductive Medicine, 2022
In the same year, a group of UK-based scientists used a similar CRISPR-Cas9-based approach to edit a group of human zygotes in order to investigate the regulation of early human development. This study was carried out on embryos donated for research purposes and was conducted under license from the UK regulator (HFEA). In this case, the study aimed to knockout gene function rather than correct a mutation (26). The authors selected the POU5F1 gene, encoding the OCT4 protein, an interesting target given its essential role in the maintenance of pluripotency within the inner cell mass. The CRISPR-Cas9 system was employed to introduce DSBs within POU5F1 and the researchers relied upon error-prone NHEJ to create indels during the repair process, disrupting the coding region of the gene and producing premature stop codons. Using this strategy, it was shown that OCT4 is essential for the formation and maintenance of human blastocysts (26).
Embryology
Published in Anthony R. Mundy, John M. Fitzpatrick, David E. Neal, Nicholas J. R. George, The Scientific Basis of Urology, 2010
The advent of research methods such as polymerase chain reaction and fluorescent in situ hybridization have greatly facilitated the study of the genetic mechanisms regulating the complex and tightly ordered anatomical sequence that characterizes normal embryological development. As well as mapping and sequencing genes, research methodology is identifying many of the gene products responsible for implementing the genetic “program’’ encoded on DNA at a cellular and molecular level. Moreover, the function of specific genes is being extensively studied in experimentally induced null or “knockout” gene deletions in experimental rodents. Relevant examples will be cited throughout the chapter.
Rodent Autosomal Dominant Polycystic Kidney Disease Models
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Sara J. Holditch, Raphael A. Nemenoff, Katharina Hopp
Gene targeting constructs come in many flavors, dependent upon the researchers’ question, but for PKD research, three general types are of primary focus: knockout, knockin, and conditional/inducible mutagenesis constructs. Knockout gene targeting constructs typically contain exon–intron sequences homologous to the endogenous gene, where one or multiple exons are replaced by a positive selection cassette ending in a stop codon (Figure 10.1a). Successful homologous recombination will result in premature transcriptional termination of their targeted allele and mimic a loss-of-function allele. Classical knockout alleles of Pkd1 or Pkd2 are briefly discussed in Section 10.4. However, classical knockout models of Pkd1 or Pkd2 have not aided tremendously in our understanding of ADPKD pathomechanisms because of their embryonic lethality in homozygosity and their very mild phenotype in heterozygosity. However, in some instances, the endogenous exon is not only replaced with a positive selection cassette but also with a reporter gene transcribed in place of the endogenous gene (Figure 10.1b). Models such as these are insightful in that they allow for characterization of endogenous gene expression patterns by assessing reporter gene expression through various staining techniques. In the case of ADPKD/ARPKD, reporter mice are available for Pkd1,41,42Pkd2,43 and Pkhd1,44 which have provided valuable insight about where in the kidney or body these genes are expressed and where their function may play an important role.
Hepatic stellate cell reprogramming via exosome-mediated CRISPR/dCas9-VP64 delivery
Published in Drug Delivery, 2021
Nianan Luo, Jiangbin Li, Yafeng Chen, Yan Xu, Yu Wei, Jianguo Lu, Rui Dong
Gene therapy is to introduce exogenous normal genes into human target cells in a certain way to correct or compensate for diseases caused by gene defects and abnormalities, so as to achieve the goal of treatment (Yla-Herttuala, 2017). Gene editing technology is a precise site-specific modification technology for genome, which can knock out, add and replace specific DNA fragments, so as to carry out precise gene editing at the genome level. This technology has become a powerful tool for gene function, genomics, gene therapy and other aspects. CRISPR/dCas9 gene editing technology is based on the bacteria or the archaea CRISPR mediated the acquired immune system derived a new type of gene editing techniques, identified by a complementary RNA by base pairing of DNA, guiding Cas9 nuclease cutting identification of double-stranded DNA, induce homologous recombination or non-homologous end link, achieve the purpose of DNA for editing (Lin et al., 2018). Compared with the traditional gene editing system, this system has the advantages of more efficient, simple operation and low cytotoxicity, and has become the most widely used genome editing tool. Currently, CRISPR/dCas9 gene editing technology has been applied in many aspects of disease research, including research on gene function, gene resistance, and building animal models (Chen et al., 2019). The CRISPR/dCas9 system can achieve efficient gene knockout, gene knockin, gene upregulation and gene downregulation, etc. However, there are still some other challenges for the application of CRISPR/dCas9 system in clinical treatment, such as the off-target effect and low delivery efficiency (Li et al., 2019).
Management of hyperuricemia and gout in obese patients undergoing bariatric surgery
Published in Postgraduate Medicine, 2018
Claudio Tana, Luca Busetto, Angelo Di Vincenzo, Fabrizio Ricci, Andrea Ticinesi, Fulvio Lauretani, Antonio Nouvenne, Maria Adele Giamberardino, Francesco Cipollone, Roberto Vettor, Tiziana Meschi
In a minor quote, UA accumulates from a reduced intestinal excretion. In the gastrointestinal (GI) tract, UA derives from circulation, bile, saliva, and peptic juices and is usually completely reabsorbed by intestinal transporters or degraded [22]. Knockout gene of the urate transporter ABCG2, represented both in the kidney and in the GI tract, is associated with elevated serum levels of UA due to a reduced intestinal excretion [23–25]. ABCG2 variants were associated with hyperuricemia also in overweight patients with type 2 diabetes [26]. Despite the evidence is actually limited, these data may suggest a possible inherited predisposition to gout of obese patients.
FTO Gene Affects Obesity and Breast Cancer Through Similar Mechanisms: A New Insight into the Molecular Therapeutic Targets
Published in Nutrition and Cancer, 2018
M. E. Akbari, M. Gholamalizadeh, S. Doaei, F. Mirsafa
The influence of the IRX3 gene on body weight and body composition is well known (48-50). In mice with IRX3 knockout gene, body weight was decreased about 25 to 30% and the adipose tissue and adipocytes size was reduced (24). Furthermore, Yang et al suggested that IRX3 gene is involved in human breast cancer (49). These findings indicate a complex relationship between FTO gene, IRX3 gene and related health outcomes. A summary of studies description is presented in Table 1.