scholarly journals RNAi for Plant Functional Genomics

2004 ◽  
Vol 5 (3) ◽  
pp. 240-244 ◽  
Author(s):  
Louisa Matthew
Keyword(s):  
Functional Genomics ◽  
Insertional Mutagenesis ◽  
Plant Biology ◽  
Plant Genome ◽  
Specific Gene ◽  
Hairpin Rna ◽  
Knock Out ◽  
Gene Redundancy ◽  
Plant Genes ◽  
Straightforward Approach

A major challenge in the post-genome era of plant biology is to determine the functions of all the genes in the plant genome. A straightforward approach to this problem is to reduce or knock out expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study, but it is limited by gene redundancy, lethal knock-outs, nontagged mutants and the inability to target the inserted element to a specific gene. RNA interference (RNAi) of plant genes, using constructs encoding self-complementary ‘hairpin’ RNA, largely overcomes these problems. RNAi has been used very effectively inCaenorhabditis elegansfunctional genomics, and resources are currently being developed for the application of RNAi to high-throughput plant functional genomics.

High-throughput vectors for efficient gene silencing in plants

2002 ◽  
Vol 29 (10) ◽  
pp. 1217 ◽  
Author(s):  
Chris A. Helliwell ◽  
S. Varsha Wesley ◽  
Anna J. Wielopolska ◽  
Peter M. Waterhouse
Keyword(s):  
Gene Silencing ◽  
Insertional Mutagenesis ◽  
Single Step ◽  
Plant Genome ◽  
Specific Gene ◽  
Single Polymerase Chain Reaction ◽  
Efficient Gene ◽  
Gene Redundancy ◽  
Endogenous Genes ◽  
Rapid Generation

A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs. pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.

scholarly journals Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuu Asano ◽  
Kensuke Yamashita ◽  
Aoi Hasegawa ◽  
Takanori Ogasawara ◽  
Hoshie Iriki ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dictyostelium Discoideum ◽  
Streptococcus Pyogenes ◽  
Nucleotide Substitution ◽  
Point Mutations ◽  
Targeted Mutagenesis ◽  
Single Amino Acid ◽  
Specific Gene ◽  
Knock Out ◽  
Protospacer Adjacent Motif

AbstractThe powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

Serial analysis of gene expression (SAGE) during porcine embryo development

2004 ◽  
Vol 16 (2) ◽  
pp. 87 ◽  
Author(s):  
Le Ann Blomberg ◽  
Kurt A. Zuelke
Keyword(s):  
Gene Expression ◽  
Functional Genomics ◽  
Embryo Development ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Transgenic Animals ◽  
Specific Gene ◽  
Research Strategies

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

Rice Functional Genomics by Transposon Mutagenesis

2002 ◽  
Vol 06 (24) ◽  
pp. 930-935 ◽  
Author(s):  
Chang-deok Han
Keyword(s):  
Tissue Culture ◽  
Transposable Elements ◽  
Functional Genomics ◽  
Large Scale ◽  
Expression Patterns ◽  
Transposon Mutagenesis ◽  
Transposon Tagging ◽  
Genomic Sequences ◽  
Knock Out ◽  
Genetic Cross

Transposable elements are powerful mutagens. Along with genomic sequences, knock-out phenotypes and expression patterns are important information to elucidate the function of genes. In this review, I propose a strategy to develop tranposant lines on a large scale by combining genetic cross and tissue culture of Ac and Ds lines. Based on the facts that Ds tends to be inactive in F2 or later generation and Ds becomes reactivated via tissue culture, a large scale of transposants can be produced by tissue culture of seeds carrying Ac and inactive Ds. In this review, I describe limitations and considerations in operating transposon tagging systems in rice. Also, I discuss the efficiency of our gene trap system and technical procedures to clone Ds flanking DNA.

Insertional mutagenesis: a Swiss Army knife for functional genomics of Medicago truncatula

2005 ◽  
Vol 10 (5) ◽  
pp. 229-235 ◽  
Author(s):  
Million Tadege ◽  
Pascal Ratet ◽  
Kirankumar S. Mysore
Keyword(s):  
Functional Genomics ◽  
Medicago Truncatula ◽  
Insertional Mutagenesis

scholarly journals Red flour beetle (Tribolium castaneum): From population genetics to functional genomics

2018 ◽  
pp. 1043-1046 ◽  
Author(s):  
Harshit Kumar ◽  
Manjit Panigrahi ◽  
Supriya Chhotaray ◽  
V. Bhanuprakash ◽  
Rahul Shandilya ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Tribolium Castaneum ◽  
Insertional Mutagenesis ◽  
Molecular Genetic ◽  
Genetic Studies ◽  
Immune Priming ◽  
Systemic Rnai ◽  
Basic Facts

Tribolium castaneum is a small and low maintenance beetle that has emerged as a most suitable insect model for studying developmental biology and functional genetic analysis. Diverse population genetic studies have been conducted using Tribolium as the principal model to establish basic facts and principles of inbreeding experiments and response to the selection and other quantitative genetics fundamentals. The advanced molecular genetic studies presently focused on the use of Tribolium as a typical invertebrate model for higher diploid eukaryotes. After a whole genome sequencing of Tribolium, many areas of functional genomics were unraveled, which enabled the use of it in many technical approaches of genomics. The present text reviews the use of Tribolium in techniques such as RNAi, transgenic studies, immune priming, immunohistochemistry, in situ hybridization, gene sequencing for characterization of microRNAs, and gene editing using engineered endonuclease. In contrast to Drosophila, the T. castaneum holds a robust systemic RNAi response, which makes it an excellent model for comparative functional genetic studies. Keywords: functional genomics, hox gene, insertional mutagenesis, RNAi, Tribolium.

scholarly journals Evolutionary Dynamics of Transposable Elements Following a Shared Polyploidization Event in the Tribe Andropogoneae

2020 ◽  
Vol 10 (12) ◽  
pp. 4387-4398
Author(s):  
Dhanushya Ramachandran ◽  
Michael R. McKain ◽  
Elizabeth A. Kellogg ◽  
Jennifer S. Hawkins
Keyword(s):  
Insertional Mutagenesis ◽  
Evolutionary Dynamics ◽  
Diploid Species ◽  
Duplicate Gene ◽  
Plant Genome ◽  
Abiotic Stress Response ◽  
Repeat Content ◽  
Gene Copies ◽  
Polyploidization Event ◽  
Plant Genome Evolution

Both polyploidization and transposable element (TE) activity are known to be major drivers of plant genome evolution. Here, we utilize the Zea-Tripsacum clade to investigate TE activity and accumulation after a shared polyploidization event. Comparisons of TE evolutionary dynamics in various Zea and Tripsacum species, in addition to two closely related diploid species, Urelytrum digitatum and Sorghum bicolor, revealed variation in repeat content among all taxa included in the study. The repeat composition of Urelytrum is more similar to that of Zea and Tripsacum compared to Sorghum, despite the similarity in genome size with the latter. Although LTR-retrotransposons were abundant in all species, we observed an expansion of the copia superfamily, specifically in Z. mays and T. dactyloides, species that have adapted to more temperate environments. Additional analyses of the genomic distribution of these retroelements provided evidence of biased insertions near genes involved in various biological processes including plant development, defense, and macromolecule biosynthesis. Specifically, copia insertions in Zea and T. dactyloides were significantly enriched near genes involved in abiotic stress response, suggesting independent evolution post Zea-Tripsacum divergence. The lack of copia insertions near the orthologous genes in S. bicolor suggests that duplicate gene copies generated during polyploidization may offer novel neutral sites for TEs to insert, thereby providing an avenue for subfunctionalization via TE insertional mutagenesis.

scholarly journals Reverse Genetics for Functional Genomics of Phytopathogenic Fungi and Oomycetes

2009 ◽  
Vol 2009 ◽  
pp. 1-11 ◽  
Author(s):  
Vijai Bhadauria ◽  
Sabine Banniza ◽  
Yangdou Wei ◽  
You-Liang Peng
Keyword(s):  
Functional Genomics ◽  
Gene Disruption ◽  
Insertional Mutagenesis ◽  
Reverse Genetics ◽  
Phytopathogenic Fungi ◽  
Sequence Information ◽  
Biological Functions ◽  
Targeted Gene Disruption ◽  
Local Lesions ◽  
Genome Sequence Information

Sequencing of over 40 fungal and oomycete genomes has been completed. The next major challenge in modern fungal/oomycete biology is now to translate this plethora of genome sequence information into biological functions. Reverse genetics has emerged as a seminal tool for functional genomics investigations. Techniques utilized for reverse genetics like targeted gene disruption/replacement, gene silencing, insertional mutagenesis, and targeting induced local lesions in genomes will contribute greatly to the understanding of gene function of fungal and oomycete pathogens. This paper provides an overview on high-throughput reverse genetics approaches to decode fungal/oomycete genomes.

Rice Gene Machine: A Vehicle for Finding Functions of Cereal Genes

2002 ◽  
Vol 06 (24) ◽  
pp. 936-942 ◽  
Author(s):  
Narayana M. Upadhyaya ◽  
Qianhao Zhu ◽  
Andrew Eamens ◽  
Elizabeth S. Dennis
Keyword(s):  
Insertional Mutagenesis ◽  
Genomic Research ◽  
Gene Control ◽  
Plant Industry ◽  
Asian Countries ◽  
Rice Gene ◽  
Production Constraints ◽  
Plant Genes ◽  
Insertion Sites ◽  
To Come

Identifying functions for the predicted 25 000 to 40 000 plant genes is the new focus of genomic research following the sequencing of genomes from model plants like Arabidopsis and rice. It is important that researchers and plant breeders especially from Asian countries access the techniques and resources of plant functional genomics, not only to remain competitive globally in rice research and development activities, but also to increase yields by alleviating some of the rice production constraints. These newly identified genes and gene control sequences can be used in transformation breeding and as molecular markers in classical plant breeding. At CSIRO Plant Industry we are using an insertional mutagenesis approach in rice to relate genes to their functions. With national and international collaboration we hope to create a substantial "Rice Gene Machine" comprised of many thousands of rice insertion lines with their associated phenotypes and the gene sequences flanking insertion sites identified. This Rice Gene Machine will allow the identification of gene functions, which will benefit rice research and the rice industry for years to come.

Hematopoietic Specific GATA-1-Improved Cre Mouse for Erythroid-Specific Gene Modification.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1291-1291
Author(s):  
Donghoon Yoon ◽  
Bumjun Kim ◽  
Myunghi Kwon ◽  
Josef T. Prchal
Keyword(s):  
Bone Marrow ◽  
Transgenic Mouse ◽  
Erythropoietin Receptor ◽  
Specific Gene ◽  
Hematopoietic Tissue ◽  
Knock Out ◽  
Tissue Sections ◽  
Lacz Staining ◽  
Splenic Cells ◽  
Beta Galactosidase

Abstract Animal models of erythropoiesis related genes have been limited by the fact that some of these genes have non-erythroid expression and other functions in addition to erythropoiesis and thus their knock-out may be embryonic lethal. Tissue specific knock-out or knock-in mice models employing GATA-1-Cre and other constructs showed that these promoters are also active in non-hematopoietic tissues, i.e. GATA-1 has activity in early embryonic development and in neuronal tissue. Suzuki et al (Blood, 2002, 100; 2279) isolated the GATA-1 locus hematopoietic regulatory domain (GATA-1-HRD) and demonstrated that the expression of a transgene under its control is limited to the hematopoietic tissue. We generated a transgenic mouse expressing an improved Cre (iCre) under GATA-1-HRD promoter control. This mouse was crossbred with ROSA 26 mouse and the progeny was examined for tissue specificity of iCre expression using beta-galactosidase staining. Brain, spleen, kidney, heart, thymus, liver, lung and ovary were examined for whole organ LacZ staining. All tested organs were negative except kidney and spleen where some positivity was observed. Subsequently, we prepared tissue sections from kidney, spleen and bone marrow and stained with LacZ and anti-beta-galactosidase antibody. Only the bone marrow EpoR expressing cells were positive; the kidney and the spleen cells were negative. Although Suzuki et al previously showed expression of the GATA-1-HRD driven erythropoietin receptor in spleen using RT-PCR, we were not able to find iCre expression in the splenic cells using these approaches. We demonstrate that our transgenic mouse (GATA-1-HRD-iCre) showed a restricted iCre expression in hematopoietic tissue that differs from previous studies of other hematopoiesis specific cre mouse. We conclude that this mouse model should be useful in studies of function of erythroid specific genes.

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