Model System For Genetic Analysis Research Articles

Zebrafish Embryonic Slow Muscle Is a Rapid System for Genetic Analysis of Sarcomere Organization by CRISPR/Cas9, but Not NgAgo.

Zebrafish embryonic slow muscle cells, with their superficial localization and clear sarcomere organization, provide a useful model system for genetic analysis of muscle cell differentiation and sarcomere assembly. To develop a quick assay for testing CRISPR-mediated gene editing in slow muscles of zebrafish embryos, we targeted a red fluorescence protein (RFP) reporter gene specifically expressed in slow muscles of myomesin-3-RFP (Myom3-RFP) zebrafish embryos. We demonstrated that microinjection of RFP-sgRNA with Cas9 protein or Cas9 mRNA resulted in a mosaic pattern in loss of RFP expression in slow muscle fibers of the injected zebrafish embryos. To uncover gene functions in sarcomere organization, we targeted two endogenous genes, slow myosin heavy chain-1 (smyhc1) and heat shock protein 90 α1 (hsp90α1), which are specifically expressed in zebrafish muscle cells. We demonstrated that injection of Cas9 protein or mRNA with respective sgRNAs targeted to smyhc1 or hsp90a1 resulted in a mosaic pattern of myosin thick filament disruption in slow myofibers of the injected zebrafish embryos. Moreover, Myom3-RFP expression and M-line localization were also abolished in these defective myofibers. Given that zebrafish embryonic slow muscles are a rapid in vivo system for testing genome editing and uncovering gene functions in muscle cell differentiation, we investigated whether microinjection of Natronobacterium gregoryi Argonaute (NgAgo) system could induce genetic mutations and muscle defects in zebrafish embryos. Single-strand guide DNAs targeted to RFP, Smyhc1, or Hsp90α1 were injected with NgAgo mRNA into Myom3-RFP zebrafish embryos. Myom3-RFP expression was analyzed in the injected embryos. The results showed that, in contrast to the CRISPR/Cas9 system, injection of the NgAgo-gDNA system did not affect Myom3-RFP expression and sarcomere organization in myofibers of the injected embryos. Sequence analysis failed to detect genetic mutations at the target genes. Together, our studies demonstrate that zebrafish embryonic slow muscle is a rapid model for testing gene editing technologies in vivo and uncovering gene functions in muscle cell differentiation.

The translational relevance of Drosophila in drug discovery

After nearly a century of successful research, the value of the fly Drosophila melanogaster as a model system for genetic analysis is well established. In the past few years, Drosophila has also successfully been used for living‐organism‐based chemical screenings [1], [2]. Because it is still a new tool in this field, the value of flies for drug discovery is still under assessment and one of the recurrent and fair questions that remains open regards whether or not it is likely that compounds identified through screenings in flies are also going to be functional in mammals. To contribute to an answer, we have inverted the question and searched the literature for compounds that were originally known for their activity in human cells and were later shown to have the same molecular mechanism of action in Drosophila . We found that such compounds are not rare. Table EV1 shows a selection of 60 such compounds, including actin and microtubule poisons; inhibitors of DNA topoisomerases, kinases, and phosphatases; alkylating agents; …

Vascular anatomy of the developing medaka, Oryzias latipes: A complementary fish model for cardiovascular research on vertebrates

The zebrafish has become a very useful vertebrate model for cardiovascular research, but detailed morphogenetic studies have revealed that it differs from mammals in certain aspects of the primary circulatory system, in particular, the early vitelline circulation. We searched for another teleost species that might serve as a complementary model for the formation of these early primary vessels. Here (and online at http://www.shigen.nig.ac.jp/medaka/atlas/), we present a detailed characterization of the vascular anatomy of the developing medaka embryo from the stage 24 (1 day 20 hr) through stage 30 (3 days 10 hr). Three-dimensional images using confocal microangiography show that the medaka, Oryzias latipes, follows the common embryonic circulatory pattern consisting of ventral aorta, aortic arches, dorsal aorta, transverse vessels, vitelline capillary plexus, and marginal veins. The medaka, thus, may serve as a valuable model system for genetic analysis of the primary vasculature of vertebrates.

Wsh3/Tea4 Is a Novel Cell-End Factor Essential for Bipolar Distribution of Tea1 and Protects Cell Polarity under Environmental Stress in S. pombe

The fission yeast Schizosaccharomyces pombe has a cylindrical cell shape, for which growth is strictly limited to both ends, and serves as an excellent model system for genetic analysis of cell-polarity determination. Previous studies identified a cell-end marker protein, Tea1, that is transported by cytoplasmic microtubules to cell tips and recruits other cell-end factors, including the Dyrk-family Pom1 kinase. The deltatea1 mutant cells cannot grow in a bipolar fashion and show T-shaped morphology after heat shock. We identified Wsh3/Tea4 as a novel protein that interacts with Win1 MAP kinase kinase kinase (MAPKKK) of the stress-activated MAP kinase cascade. Wsh3 forms a complex with Tea1 and is transported to cell tips by growing microtubules. The deltawsh3 mutant shows monopolar growth with abnormal Tea1 aggregate at the non-growing cell end; this abnormal aggregate fails to recruit Pom1 kinase. Consistent with the observed interaction between Win1 and Wsh3, cells lacking Wsh3 or Tea1 show more severe cell-polarity defects under osmolarity and heat-stress stimuli that are known to activate the stress MAPK cascade. Furthermore, mutants of the stress MAPK also exhibit cell-polarity defects when exposed to the same stress. Wsh3/Tea4 is an essential component of the Tea1 cell-end complex. In addition to its role in bipolar growth during the normal cell cycle, the Wsh3-Tea1 complex, together with the stress-signaling MAPK cascade, contributes to cell-polarity maintenance under stress conditions.

Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells.

The ability to generate mutations is a prerequisite to functional genetic analysis. Despite a long history of using mice as a model system for genetic analysis, the scientific community has not generated a comprehensive collection of multiple alleles for most mouse genes. The chemical mutagen of choice for mouse has been N-ethyl-N-nitrosourea (ENU), an alkylating agent that mainly causes base substitutions in DNA, and therefore allows for recovery of complete and partial loss-, as well as gain-, of-function alleles . Specific locus tests designed to detect recessive mutations showed that ENU is the most efficient mutagen in mouse with an approximate mutation rate of 1 in 1,000 gametes. In fact, several genome-wide and region-specific screens based on phenotypes have been carried out. The anticipation of the completion of the human and mouse genome projects, however, now emphasizes genotype-driven genetics--from sequence to mutants. To take advantage of the mutagenicity of ENU and its ability to create allelic series of mutations, we have developed a complementary approach to generating mutations using mouse embryonic stem (ES) cells. We show that a high mutation frequency can be achieved and that modulating DNA-repair activities can enhance this frequency. The treated cells retain germline competency, thereby rendering this approach applicable for efficient generation of an allelic series of mutations pivotal to a fine-tuned dissection of biological pathways.

Restriction endonuclease mapping and cloning of Mycobacterium intracellulare plasmid pLR7

A restriction map of Mycobacterium intracellulare plasmid pLR7 was developed. This 15.3-kb plasmid had unique sites for BamHI, HindIII, and XbaI. Various large fragments of pLR7 were cloned into pBR322 or pHP34 and propagated in Escherichia coli. A hybrid pLR7:: pBR322 plasmid carrying the complete pLR7 sequence was constructed by joining the plasmids at their HindIII sites. The construction of these hybrids will facilitate the analysis and manipulation of pLR7 and may allow the development of this plasmid as a model system for genetic analysis in mycobacteria.

Embryo-lethal mutants of Arabidopsis thaliana: A model system for genetic analysis of plant embryo development

Arabidopsis thaliana (Cruciferae) has been chosen as a model system for mutant analysis of plant embryo development. The isolation and characterization of embryo-lethal mutants of Arabidopsis are simplified by the following genetic and developmental characteristics: (1) Aborted seeds can be easily recognized in both immature and mature fruits; (2) spontaneous or physiological embryo abortion is rare; (3) individual fruits contain 30–60 seeds and show clear segregation of normal and aborted seeds in plants heterozygous for embryo-lethal mutations; (4) mature plants contain hundreds of seeds at every stage of development; (5) the generation time of 5–6 weeks is one of the shortest among flowering plants; (6) the diploid chromosome number is 10, and a large number of morphological mutants are available for linkage studies; (7) plants naturally self-pollinate but can be experimentally cross-pollinated; (8) the pattern of normal embryo development is very similar to that of Capsella, a plant used extensively for descriptive and experimental studies; (9) embryo and ovule culture can be used to study the biochemistry of mutant embryos; and (10) the isolation of temperature-sensitive mutants is possible because mature plants are small and viable over a wide range of temperatures. This report describes normal development in Arabidopsis thaliana, strain “Columbia,” and outlines the system used to identify embryo-lethal mutants. A method of classifying embryo-lethal mutants as cellular, nutritional, or developmental lethals is presented, and the potential application of these mutants to the study of normal embryo development is discussed.