Question: Diagram and describe the genetic screen in which the frozen mutant was

Question

Question: Diagram and describe the genetic screen in which the frozen mutant was isolated.

*Answer: the answer is included within attached material and results sections. Thanks!

Note: please write on your own words, diagram should be made by you, and include sources please. You can draw the diagram on a paper and attach as a picture into this post.

Additional information:

myotome and heart in the developing mouse em bryo, Aima (dpf Wild-type embryos were obtained from the AB strain. et al. 2008) wi The fro mutation that was isolated in a large-scale chem hil e in C2C12 cells, Myo18B is expressed at a basal level in undifferentiated myoblasts and upregulated upon ical mutagenesis screen at the Max-Planck Institute fur their differentiation into myofibers (Salamon et aL 2003 Entwicklungsbiologie (Granato et al. 1996) was maintained in the AB genetic background and outcrossed to the polymor Two recent studies have associated mutations of human MY018B with nemaline myopathy. In the first of these, two phic WIK (L11) strain of wild-type fish to generate mapping patients with Klippel-Feil anomaly (KFA) were found to be embryos for pos tional cloning. The Tg smyhc1:GFP) homozygous for a nonsense mutation in the penultimate transgenic line has been previously described (Elworthy et al. exon of the MY018B gene (Alazami et al 2015); notably, 2008). Fish were maintained and manipulated in the Univ sity of Sheffield and the Institute of Molecular and Cell B both patients exhibited symptoms of myopathy Chypotonia and muscle weakness, a condition not previously associated ology (IMCB) zebrafish facilities in compliance with the UK with KFA. Muscle biopsy analysis of one patient revealed Animals (Scientific Procedures Act 1986 or the Biomedica Research Council (BMRC) Institutional Animal Care and Use variation in fiber size and a loss of normal banding indicative Committee (IACUC guidelines, respectively. of a loss of thick filaments. Electron microscopy (EM) anal ysis also revealed scattered, dense bodies reminiscent of Induction of new froimyo 18b mutant alleles nemaline rods, typical of other human myopathies (Wallgren et al. In a second study, a single infant with ENU mutagenesis: Adult wild-type (AB) male zebrafish Pettersson 201 mutagenized using ENU according to an improved severe axial and peripheral hypotonia, who died at 4 months Were protocol (Kettleborough et al. 2011). The G0 was found to be homozygous for a premature termination mutagene mutagenized individuals were outcrossed to wild-type codon in the last exon of the MY018B gene. Muscle biopsies females and the resultant F1 males test-crossed to fe- from this individual revealed variation in fiber size as well as o27e males heterozygous for the fro allele. Each cross was small nemaline rods (Malfatti et al. 2015) screened for immotile progeny and a single ma segregat In contrast to the postnatal survival of the KFA patients ing an allele that failed to complement the fro 27c allel reverse genetic studies ha revealed Myo18B to be essential was identified. The allele transmitted by this male was r normal embryonic development inthe mouse (Ajima e designated fro 1230 2008). Embryos homozygous for a targeted knockout of the ocus die of cardiac defects by E10.5 and show disrupted. Clustered regularly interspaced short palindromic repeats myofibrils in their cardiomyocytes, the thick and thin fila (CRISPRDA Cas9 muti A target sequence in exon 3 of ments of the sarcomere appearing misaligned. This early em the myo18b gene (5'-GGCCGAGATG TCGCTGCGAG-3) was bryonic lethality and growth retardation of mutant embryos identified using ZiFIT Targeter software partners. (http however, precluded analysis of Myo18B function in skeletal org/ZiFiT/) and the sequence inserted into the Bsal site muscles of pDR274 guide RNA (gRNA) expression vector (Hwang Forward genetic screens for mutations affecting muscle 2013). To synthesize gRNA, the template DNA frag development and function in the zebrafish yielded a number ment was amplified from the vector using the primer pair f mutants including frozen fro) and sloth, originally (forward: 5'-CATTATGGTGAAAG TTGGAAC-3' and reverse fied on the basis of their complete lack of motility (Granato 5'-AAAAAGCACCGACTCGGTGCOAC-3') and gRNA Was tran- et al. 1996). Morphological analysis of sloth and fro embryos scribed using a MEGAshortscript T7 Kit (Invitrogen, Carlsbad, revealed an absence of striated muscle fibers and a failure of CA) following the manufacturers instructions. gRNA was muscle ce nuclei to elongate, suggestive of an early block in injected into fertilized eggs at 100 ng/jul together with their differentiation (Granato et al. 1996). Molecular charac codon-optimized Cas9 messenger RNA (mRNA with nuclear terization of the sloth locus showed the phenotype to be due localization signals at 150 ng/ul. The Cas9 mRNAwas syn to a f-function mutation in the hsp90A gene, implicating thesized from the pCS2-nCas9n vector (Jao et al. 2013) the chaperone protein that it encodes in myofibril formation Injected embryos were raised to adults and F1 embryos (Hawkins et al. 2008). Here, we show that the fro mutant screened for targeted mutations using the primer pair phenotype is caused by loss-of-function of the zebrafish ward: 5'-CCAGCAAACTCAGCGACAACCTA-3' and reverse myo18b gene. Our analysis reveals that myo18b is expressed 5'-CTG CAGGTCT GTGT TTCTGC-3') to amplify the target specifically in precurs s of fast-twitch skeletal muscle fibers locus and sequence for genotyping in which it is required for the assembly of myofibrils Positional cloning of the fro locus Materials and Methods Simple sequence length polymorphisms that flanked the fro locus, z21976 and z3260, were used to identify 69 and Zebrafish and husbandry 21 recombinants, respectively, from 1774 meioses, as pre- Zebrafish were raised, bred, and staged following standard viously described (Talbot and Schie 1999). The full methods (Kimmel et al. 1995). Developmental stages are anno- length coding sequence of zebrafish myo18b was deduced tated as hours postfertilization Chpf or days postfertilization from four ESTs (LOC100537963, ENDSTART00000122824 R. Gurung et a 726

Explanation / Answer

Induction of fro/myo18b mutant alleles:-

Wild – type AB was mutagenized using ENU

Mutagenized individuals were outcrossed to wild type females

F1 males were test crossed to females heterozygous for the froto27c allele.

A single male segregating an allele that failed to compliment the froto27c allele was identified.

Positional cloning of fro locus:-

Results of the Experiment:-

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