scholarly journals mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces potentially functional transcripts

2017 ◽  
Author(s):  
Jennifer L Anderson ◽  
Timothy S Mulligan ◽  
Meng-Chieh Shen ◽  
Hui Wang ◽  
Catherine M Scahill ◽  
...  
Keyword(s):  
Gene Function ◽  
Reverse Genetics ◽  
Model Organism ◽  
Low Frequency ◽  
Mrna Processing ◽  
Null Alleles ◽  
Reverse Genetic ◽  
Loss Of Function ◽  
Genomic Change ◽  
Frame Shift

AbstractAs model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, allow frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of genomic compensation by means of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of genomic adaptations that can occur may inform the strategies of mutant generation.Author summaryThe recent rise of reverse genetic, gene targeting methods has allowed researchers to readily generate mutations in any gene of interest with relative ease. Should these mutations have the predicted effect on the mRNA and encoded protein, we would expect many more abnormal phenotypes than are typically being seen in reverse genetic screens. Here we set out to explore some of the reasons for this discrepancy by studying seven separate mutations in zebrafish. We present evidence that thorough cDNA sequence analysis is a key step in assessing the likelihood that a given mutation will produce hypomorphic or null alleles. This study reveals that alternative mRNA processing in the mutant background often produces transcripts that escape nonsense-mediated decay, thereby potentially preserving gene function. By understanding the ways that cells avoid the deleterious consequences of mutations, researchers can better design reverse genetic strategies to increase the likelihood of gene disruption.

scholarly journals Identification and Characterization of Genes That Interact With lin-12 in Caenorhabditis elegans

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1675-1695 ◽  
Author(s):  
Frans E Tax ◽  
James H Thomas ◽  
Edwin L Ferguson ◽  
H Robert Horvitzt
Keyword(s):  
Cell Fate ◽  
Low Frequency ◽  
Signal Transduction Pathway ◽  
Temperature Shift ◽  
Egg Laying ◽  
Null Alleles ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Suppressor Mutations

Abstract We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-l7, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup1 7 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup1 7and lag-2, suggest that both genes act at approximately the same time as lin-12in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.

scholarly journals A dual sgRNA approach for functional genomics in Arabidopsis thaliana[OPEN]

2017 ◽  
Author(s):  
Laurens Pauwels ◽  
Rebecca De Clercq ◽  
Jonas Goossens ◽  
Sabrina Iñigo ◽  
Clara Williams ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Gene Editing ◽  
Reverse Genetics ◽  
Gene Families ◽  
Null Alleles ◽  
Loss Of Function ◽  
Knock Out ◽  
Nonsense Mediated Decay ◽  
Dna Insertion ◽  
Entire Gene

AbstractReverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows to generate knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain Cas9 null-segregants with bi-allelic mutations in the T2 generation. Out of seven new mutant alleles we report here, one allele for GRXS17, the ortholog of human GRX3/PICOT, did not phenocopy previously characterized nulls. Notwithstanding, the mutation caused a frameshift and triggered nonsense-mediated decay. We demonstrate that our workflow is also compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method can result in a more reliable loss-of-function alleles that lack a large essential part of the gene. The ease in the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.One-sentence summaryWe present a dual sgRNA approach to delete Arabidopsis gene 34 fragments in order to obtain reliable functional knock-outs.

What Makes a Weed a Weed? How Virus-mediated Reverse Genetics Can Help to Explore the Genetics of Weediness

2020 ◽  
Vol 31 (5) ◽  
pp. 224-229
Author(s):  
Dana R. MacGregor
Keyword(s):  
Gene Function ◽  
Gene Networks ◽  
Reverse Genetics ◽  
Specific Gene ◽  
Reverse Genetic ◽  
Virus Induced Gene Silencing ◽  
Weed Species ◽  
Genetic Techniques ◽  
Agricultural Weeds ◽  
Insight Into

Reverse genetics investigates what a gene does by testing how the plant responds when the specific gene is changed. These techniques have been in use for decades to assess whether a given gene underpins interesting phenotypes and gain insight into the function of gene networks and families. Weed science has only recently entered the "genomic era" in which genomic and reverse genetics approaches are used to address hypotheses. This review focuses on two reverse genetic techniques used on a variety of plants including agricultural weeds, virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX), explaining the biology behind them and highlighting how these tools may be used for gene function validation in weed species for which no other transgenic approaches have been developed.

Reverse genetics in zebrafish

2000 ◽  
Vol 2 (2) ◽  
pp. 37-48 ◽  
Author(s):  
ARNE C. LEKVEN ◽  
KATHRYN ANN HELDE ◽  
CHRISTOPHER J. THORPE ◽  
REBECCA ROOKE ◽  
RANDALL T. MOON
Keyword(s):  
Reverse Genetics ◽  
Molecular Genetic ◽  
Point Mutations ◽  
Model System ◽  
Reverse Genetic ◽  
Genetic Approach ◽  
Loss Of Function ◽  
Complementation Groups ◽  
Reverse Genetic Approach ◽  
Subsequent Identification

Lekven, Arne C., Kathryn Ann Helde, Christopher J. Thorpe, Rebecca Rooke, and Randall T. Moon. Reverse genetics in zebrafish. Physiol Genomics 2: 37–48, 2000.—The zebrafish has become a popular model system for the study of vertebrate developmental biology because of its numerous strengths as a molecular genetic and embryological system. To determine the requirement for specific genes during embryogenesis, it is necessary to generate organisms carrying loss-of-function mutations. This can be accomplished in zebrafish through a reverse genetic approach. This review discusses the current techniques for generating mutations in known genes in zebrafish. These techniques include the generation of chromosomal deletions and the subsequent identification of complementation groups within deletions through noncomplementation assays. In addition, this review will discuss methods currently being evaluated that may improve the methods for finding mutations in a known sequence, including screening for randomly induced small deletions within genes and screening for randomly induced point mutations within specific genes.

scholarly journals Genotype to Phenotype: CRISPR Gene Editing Reveals Genetic Compensation as a Mechanism for Phenotypic Disjunction of Morphants and Mutants

2021 ◽  
Vol 22 (7) ◽  
pp. 3472
Author(s):  
Cristy M. Salanga ◽  
Matthew C. Salanga
Keyword(s):  
Gene Silencing ◽  
Reverse Genetics ◽  
Model Organisms ◽  
Reverse Genetic ◽  
Loss Of Function ◽  
Compensation Mechanisms ◽  
Forward Genetic Screens ◽  
Genomic Gene ◽  
Reproductive Rates ◽  
Genetic Compensation

Forward genetic screens have shown the consequences of deleterious mutations; however, they are best suited for model organisms with fast reproductive rates and large broods. Furthermore, investigators must faithfully identify changes in phenotype, even if subtle, to realize the full benefit of the screen. Reverse genetic approaches also probe genotype to phenotype relationships, except that the genetic targets are predefined. Until recently, reverse genetic approaches relied on non-genomic gene silencing or the relatively inefficient, homology-dependent gene targeting for loss-of-function generation. Fortunately, the flexibility and simplicity of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has revolutionized reverse genetics, allowing for the precise mutagenesis of virtually any gene in any organism at will. The successful integration of insertions/deletions (INDELs) and nonsense mutations that would, at face value, produce the expected loss-of-function phenotype, have been shown to have little to no effect, even if other methods of gene silencing demonstrate robust loss-of-function consequences. The disjunction between outcomes has raised important questions about our understanding of genotype to phenotype and highlights the capacity for compensation in the central dogma. This review describes recent studies in which genomic compensation appears to be at play, discusses the possible compensation mechanisms, and considers elements important for robust gene loss-of-function studies.

scholarly journals Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3669-3678 ◽  
Author(s):  
Satoshi H. Namekawa ◽  
Kazuki Iwabata ◽  
Hiroko Sugawara ◽  
Fumika N. Hamada ◽  
Akiyo Koshiyama ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Sexual Development ◽  
Reverse Genetics ◽  
Model Organism ◽  
Coprinus Cinereus ◽  
Gene Repression ◽  
Mushroom Species ◽  
Double Stranded Rna ◽  
Chromosome Synapsis ◽  
Expression Construct

The basidiomycete Coprinus cinereus has many advantages as a model organism for studying sexual development and meiosis, but it has been difficult to investigate using reverse-genetics methods, such as gene disruption by homologous recombination. Here, gene repression by dsRNA-mediated gene silencing was tried as an alternative method for reverse-genetics studies. It was shown that transformation of the LIM15/DMC1 dsRNA expression construct (LIM15dsRNA) resulted in genomic insertion of LIM15dsRNA and paucity of the LIM15/DMC1 transcript. First, LIM15dsRNA was transformed into the homothallic strain AmutBmut to generate a homozygote in which both nuclei had a copy of LIM15dsRNA. The LIM15/DMC1-repressed strain showed abnormal homologous chromosome synapsis during meiosis. Basidiospore production was reduced to 16 % by the induction of dsRNA. However, approximately 60 % of basidiospores were viable. Next, a heterozygote was generated in which one nucleus had a copy of LIM15dsRNA. The phenotype was similar to that of the homozygote. These results are not only the first demonstration of dsRNA-mediated gene silencing in a member of the homobasidiomycete fungi, to which 90 % of mushroom species belong, but also the first successful use of a reverse-genetics approach in C. cinereus research.

Genetic control of dorsal-ventral identity in the telencephalon: opposing roles for Pax6 and Gsh2

Development ◽  
2000 ◽  
Vol 127 (20) ◽  
pp. 4361-4371 ◽  
Author(s):  
H. Toresson ◽  
S.S. Potter ◽  
K. Campbell
Keyword(s):  
Cerebral Cortex ◽  
Gene Function ◽  
Abnormal Development ◽  
Loss Of Function ◽  
Embryonic Mouse ◽  
Molecular Identity ◽  
Mouse Mutants ◽  
Germinal Zone ◽  
Genetic Mechanisms ◽  
Dlx Genes

We have examined the genetic mechanisms that regulate dorsal-ventral identity in the embryonic mouse telencephalon and, in particular, the specification of progenitors in the cerebral cortex and striatum. The respective roles of Pax6 and Gsh2 in cortical and striatal development were studied in single and double loss-of-function mouse mutants. Gsh2 gene function was found to be essential to maintain the molecular identity of early striatal progenitors and in its absence the ventral telencephalic regulatory genes Mash1 and Dlx are lost from most of the striatal germinal zone. In their place, the dorsal regulators, Pax6, neurogenin 1 and neurogenin 2 are found ectopically. Conversely, Pax6 is required to maintain the correct molecular identity of cortical progenitors. In its absence, neurogenins are lost from the cortical germinal zone and Gsh2, Mash1 and Dlx genes are found ectopically. These reciprocal alterations in cortical and striatal progenitor specification lead to the abnormal development of the cortex and striatum observed in Pax6 (small eye) and Gsh2 mutants, respectively. In support of this, double homozygous mutants for Pax6 and Gsh2 exhibit significant improvements in both cortical and striatal development compared with their respective single mutants. Taken together, these results demonstrate that Pax6 and Gsh2 govern cortical and striatal development by regulating genetically opposing programs that control the expression of each other as well as the regionally expressed developmental regulators Mash1, the neurogenins and Dlx genes in telencephalic progenitors.

scholarly journals Zebrafish Cre/lox regulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation

2021 ◽  
Author(s):  
Maira P. Almeida ◽  
Sekhar Kambakam ◽  
Fang Liu ◽  
Zhitao Ming ◽  
Jordan M. Welker ◽  
...  
Keyword(s):  
Gene Function ◽  
Cre Recombinase ◽  
Gene Trap ◽  
Gene Inactivation ◽  
Loss Of Function ◽  
Cell Type ◽  
Active Gene ◽  
Indel Mutation ◽  
Targeted Integration ◽  
Cell Type Specific

The ability to regulate gene activity spatially and temporally is essential to investigate cell type specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish, but simple and efficient methods for isolation of stable, Cre/lox regulated alleles are lacking. Here we applied our GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to generate floxed conditional alleles that provide robust gene knockdown and strong loss of function phenotypes. A universal targeting vector, UFlip, with sites for cloning short 24-48 bp homology arms flanking a floxed mRFP gene trap plus secondary reporter cassette, was integrated into an intron in hdac1, rbbp4, and rb1. Active, gene off orientation hdac1-UFlip-Off and rb1-UFlip-Off integration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientation rbbp4-UFlip-On and rb1-UFlip-On integration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs of loxP and lox2272 sites, inverting and locking the cassette into the active, gene off orientation, and the expected mutant phenotypes. In combination with our endogenous neural progenitor Cre drivers we demonstrate rbbp4-UFlip-On and rb1-UFlip-On gene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP in rbbp4-UFlip-Off, or 2A-KalTA4 in rb1-UFlip-Off, shows strong RFP expression in wild type or UAS:RFP injected embryos, respectively. Together these results validate a simplified approach for efficient isolation of highly mutagenic Cre/lox responsive conditional gene alleles to advance zebrafish Cre recombinase genetics.

scholarly journals Loss-of-function in IRF2BPL is associated with neurological phenotypes

2018 ◽  
Author(s):  
Paul C. Marcogliese ◽  
Vandana Shashi ◽  
Rebecca C. Spillmann ◽  
Nicholas Stong ◽  
Jill A. Rosenfeld ◽  
...  
Keyword(s):  
Nervous System ◽  
Population Genomics ◽  
Stop Codon ◽  
Ectopic Expression ◽  
Premature Stop Codon ◽  
Model Organism ◽  
Global Developmental Delay ◽  
Mendelian Disease ◽  
Loss Of Function ◽  
Missense Variants

AbstractThe Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) gene encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals affected with neurological symptoms who carry damaging heterozygous variants in IRF2BPL. Five cases carrying nonsense variants in IRF2BPL resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The bioinformatics signature for IRF2BPL based on population genomics is consistent with a gene that is intolerant to variation. We show that the IRF2BPL ortholog in the fruit fly, called pits (protein interacting with Ttk69 and Sin3A), is broadly expressed including the nervous system. Complete loss of pits is lethal early in development, whereas partial knock-down with RNA interference in neurons leads to neurodegeneration, revealing requirement for this gene in proper neuronal function and maintenance. The nonsense variants in IRF2BPL identified in patients behave as severe loss-of-function alleles in this model organism, while ectopic expression of the missense variants leads to a range of phenotypes. Taken together, IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

scholarly journals Creating Porcine Biomedical Models Through Recombineering

2004 ◽  
Vol 5 (3) ◽  
pp. 262-267 ◽  
Author(s):  
Margarita M. Rogatcheva ◽  
Laurie A. Rund ◽  
Kelly S. Swanson ◽  
Brandy M. Marron ◽  
Jonathan E. Beever ◽  
...  
Keyword(s):  
Genetic Information ◽  
Positional Cloning ◽  
Reverse Genetics ◽  
Genetic Model ◽  
Phenotypic Diversity ◽  
Model Organism ◽  
Artificial Chromosome ◽  
Forward Genetics ◽  
Working Definition ◽  
Definition Of

Recent advances in genomics provide genetic information from humans and other mammals (mouse, rat, dog and primates) traditionally used as models as well as new candidates (pigs and cattle). In addition, linked enabling technologies, such as transgenesis and animal cloning, provide innovative ways to design and perform experiments to dissect complex biological systems. Exploitation of genomic information overcomes the traditional need to choose naturally occurring models. Thus, investigators can utilize emerging genomic knowledge and tools to create relevant animal models. This approach is referred to as reverse genetics. In contrast to ‘forward genetics’, in which gene(s) responsible for a particular phenotype are identified by positional cloning (phenotype to genotype), the ‘reverse genetics’ approach determines the function of a gene and predicts the phenotype of a cell, tissue, or organism (genotype to phenotype). The convergence of classical and reverse genetics, along with genomics, provides a working definition of a ‘genetic model’ organism (3). The recent construction of phenotypic maps defining quantitative trait loci (QTL) in various domesticated species provides insights into how allelic variations contribute to phenotypic diversity. Targeted chromosomal regions are characterized by the construction of bacterial artificial chromosome (BAC) contigs to isolate and characterize genes contributing towards phenotypic variation. Recombineering provides a powerful methodology to harvest genetic information responsible for phenotype. Linking recombineering with gene-targeted homologous recombination, coupled with nuclear transfer (NT) technology can provide ‘clones’ of genetically modified animals.

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