Approaches to Studying Polycystic Kidney Disease in Zebrafish
Jinghua Hu, Yong Yu in Polycystic Kidney Disease, 2019
Reverse genetics, including knockdown and knockout, are widely used in studying the function of genes. In the zebrafish field, as the RNAi technique is not working, we use morpholinos (MO), which are DNA analogues and knockdown gene function by blocking translation or splicing of the target transcripts. Although this technique has been recently replaced by the CRISPR/Cas9-mediated gene knockout technique, due to the off-target issue by the MO, it is still widely used. For example, if we want to block the maternal deposit of a gene transcript and the maternal-zygotic mutant is difficult to obtain, we use MO. To avoid the nonspecific effects of the MO, we should follow the guidelines.20 Basically, the criteria for a reliable MO are that the phenotypes caused by MO knockdown should phenocopy the mutants and can be rescued by its mRNA overexpression.
Gene Targeting Models of Epilepsy: Technical and Analytical Considerations
Steven L. Peterson, Timothy E. Albertson in Neuropharmacology Methods in Epilepsy Research, 2019
Given estimates that 30,000 genes are expressed in the mammalian brain,21 it is likely that the proliferation of new mutant mouse strains will include many that are relevant to the epilepsies. The first members of this new wave of epilepsy models illustrate the wide variety of genes that participate in the regulation of neuronal network excitability. It is likely that, in the future, an abundance of mechanisms involved in the regulation of excitability will be discovered. An advantage of pursuing this work in gene knockout models is that the genetic lesions are known, providing molecular points of reference for these studies. Furthermore, these models will provide candidate genes for studies aimed at uncovering the genetic bases of seizure susceptibility in humans.
Experimental perturbations to investigate cardiovascular physiology
Neil Herring, David J. Paterson in Levick's Introduction to Cardiovascular Physiology, 2018
Some of the issues with gene knockout models can be addressed with tissue- and time-specific knockouts. Tissue- specific knockout is achieved by incorporating locus of X-over P1 (loxP) sites either side of the gene of interest (floxed gene) using homologous recombination as described earlier. LoxP sites direct an enzyme called Cre recombinase that cuts and recombines sequences of DNA at these sites, thereby removing the sequence of DNA between them. The efficiency of the recombination tends to be lower the longer the length of DNA between the two loxP sites. The activity of Cre recom- binase can then be controlled by crossing the mouse with the floxed gene with another mouse expressing Cre recom- binase with a site- or cell-specific promoter as demonstrated in Figure 20.4. Examples of site/tissue-specific promoters with which Cre recombinase can be coupled include: synapsin (neuronal); PRSx8 (catecholaminergic neurons); alpha-myosin heav y chain (MHC; cardiac myocytes); smooth muscle MHC (smooth muscle); or tyrosine-protein kinase receptor Tie-2 (endothelial cells).
A two-decade journey in identifying high mobility group box 1 (HMGB1) and procathepsin L (pCTS-L) as potential therapeutic targets for sepsis
Published in Expert Opinion on Therapeutic Targets, 2023
Jianhua Li, Cassie Shu Zhu, Li He, Xiaoling Qiang, Weiqiang Chen, Haichao Wang
Even though gene knockout strategies are widely used to elucidate potential roles of various signaling molecules in many diseases, cautions should always be exercised when using these genetic approaches to evaluate extracellular roles of various inflammatory mediators. For example, despite the aforementioned pathogenic role of HMGB1 in lethal sepsis [44], genetic disruption of HMGB1 expression unexpectedly renders animals more susceptible to both infections [116] and injuries [117], suggesting distinct roles of intracellular and extracellular HMGB1 in health and disease conditions [118]. Accordingly, we generated polyclonal antibodies against murine pCTS-L and used these pharmacological agents to further characterize the extracellular role of pCTS-L in experimental sepsis (Table 3). Anti-murine pCTS-L total IgGs (pAbs) conferred dose-dependent and significant protections against lethal sepsis when the first dose was given at 2 h post CLP (Table 3) [17]. Consistently, pCTS-L antigen affinity-purified IgGs of protective pAbs effectively abrogated the pCTS-L-induced production of both TLR4-dependent cytokines (e.g. IL-6) and chemokines (e.g. MIP-1γ, LIX, RANTES, and MCP-1), as well as the RAGE-dependent neutrophilic chemokines (such as KC/GRO-α and MIP-2/GRO-β). These findings have suggested that anti-pCTS-L pAbs conferred protection against lethal sepsis possibly by attenuating pCTS-L-induced hyperinflammation likely orchestrated by both TLR4 and RAGE receptors (Figure 1).
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
Challenges with ZFNs include design and engineering of the ZFP for high-affinity binding of the desired sequence, which can prove non-trivial (Ramirez et al., 2008). Also, not all sequences are available for ZFP binding, so site selection is limited. Using open-source ZFP domains, sites could be targeted only every 200-bps in a random DNA sequence (Gupta & Musunuru, 2014). This may not be a concern if the objective is gene knockout or deletion; however, this may be an obstacle if the objective is a gene correction or addition product. Another significant challenge is off-target cutting (Gabriel et al., 2011; Pattanayak et al., 2011). ZFN design improvements addressing off-target concerns have included ZFNs that work in pairs, with each pair recognizing two sequences that flank the target cleavage site. One ZFN binds the forward strand, and the second ZFN binds the reverse strand, increasing the total number of recognized bps to between 18 and 36. Further, FokI domains that are obligate heterodimers with opposite charge have been fused to ZFPs such that only properly paired ZFNs will result in FokI dimerization/activity and the formation of a DSB (see Figure 3(A)) (Miller et al., 2007; Doyon et al., 2011).
Genome-wide CRISPR screens for the identification of therapeutic targets for cancer treatment
Published in Expert Opinion on Therapeutic Targets, 2020
Vivian Weiwen Xue, Sze Chuen Cesar Wong, William Chi Shing Cho
What is more encouraging is that new platforms and toolkits are constantly emerging in this vibrant field. The CRISPR-STOP system recently has been introduced as a more effective gene knockout tool with diminished deleterious effects compared with wild-type Cas9-mediated gene editing at high-copy-number genomic regions. This system created early stop codons and achieved subsequent silencing [106]. Besides, Kurata et al. introduced a miRNA targeted gRNA library and related CRISPR screening. This expands the application of pooled gRNA CRISPR-Cas9 screening from coding genes and lncRNAs to miRNAs [107]. Moreover, a recent study performed CRISPR screening at the single-cell level [108]. CRISPR screening in single-cell level can assist the therapeutic target discovery in individual cells or different cell populations, which helps understand tumor heterogeneity and also provides an opportunity to study tumor-associated immune cells and TME [109]. Besides, CRISPR toolkit can perform nucleic acid detection. For example, Wang et al. described an instrument-free method for rapid and sensitive African swine fever virus detection via the application of CRISPR-Cpf1 in lateral flow detection, which provided detection for samples with more than 20 viral DNA copies [110]. As a high sensitivity nucleic acid detection tool, we expect the application of CRISPR-Cpf1 in the future cancer diagnosis by detecting tumor-specific DNA.
Related Knowledge Centers
- Allele
- DNA Sequencing
- Gene Targeting
- Homologous Recombination
- Mutation
- Phenotype
- Zygosity
- Crispr Gene Editing
- Transcription Activator-Like Effector Nuclease
- Gene Knock-In