Trends in large-scale mouse mutagenesis: from genetics to functional genomics

We bring you a report from the CSHL Genome Sequencing and Biology Meeting, which has a long and prestigious history. This year there were sessions on large-scale sequencing and analysis, polymorphisms (covering discovery and technologies and mapping and analysis), comparative genomics of mammalian and model organism genomes, functional genomics and bioinformatics.

Download Full-text

The large-scale Munich ENU-mouse-mutagenesis screen

Mammalian Genome ◽

10.1007/s003350010097 ◽

2000 ◽

Vol 11 (7) ◽

pp. 507-510 ◽

Cited By ~ 26

Author(s):

Dian Soewarto ◽

Christiane Fella ◽

Andreas Teubner ◽

Birgit Rathkolb ◽

Walter Pargent ◽

...

Keyword(s):

Large Scale ◽

Mutagenesis Screen ◽

Mouse Mutagenesis

Download Full-text

Rice Functional Genomics by Transposon Mutagenesis

Asia-Pacific Biotech News ◽

10.1142/s0219030302001933 ◽

2002 ◽

Vol 06 (24) ◽

pp. 930-935 ◽

Cited By ~ 8

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.

Download Full-text

Colony size measurement of the yeast gene deletion strains for functional genomics

10.32920/14640228 ◽

2021 ◽

Author(s):

Negar Memarian ◽

Matthew Jessulat ◽

Javad Alirezaie ◽

Nadereh Mir-Rashed ◽

Jianhua Xu ◽

...

Keyword(s):

Functional Genomics ◽

Colony Size ◽

Large Scale ◽

Model Organism ◽

Antifungal Compound ◽

Image Analysis System ◽

Yeast Saccharomyces Cerevisiae ◽

Inspection Method ◽

Manual Inspection ◽

Growth Differences

Background Numerous functional genomics approaches have been developed to study the model organism yeast, Saccharomyces cerevisiae, with the aim of systematically understanding the biology of the cell. Some of these techniques are based on yeast growth differences under different conditions, such as those generated by gene mutations, chemicals or both. Manual inspection of the yeast colonies that are grown under different conditions is often used as a method to detect such growth differences. Results Here, we developed a computerized image analysis system called Growth Detector (GD), to automatically acquire quantitative and comparative information for yeast colony growth. GD offers great convenience and accuracy over the currently used manual growth measurement method. It distinguishes true yeast colonies in a digital image and provides an accurate coordinate oriented map of the colony areas. Some post-processing calculations are also conducted. Using GD, we successfully detected a genetic linkage between the molecular activity of the plant-derived antifungal compound berberine and gene expression components, among other cellular processes. A novel association for the yeast mek1 gene with DNA damage repair was also identified by GD and confirmed by a plasmid repair assay. The results demonstrate the usefulness of GD for yeast functional genomics research. Conclusion GD offers significant improvement over the manual inspection method to detect relative yeast colony size differences. The speed and accuracy associated with GD makes it an ideal choice for large-scale functional genomics investigations.

Download Full-text

Phenotypic Screen and Transcriptomics Approach Complement Each Other in Functional Genomics of Defensive Stink Gland Physiology

10.21203/rs.3.rs-1117784/v1 ◽

2021 ◽

Author(s):

Sabrina Lehmann ◽

Bibi Atika ◽

Daniela Grossmann ◽

Christian Schmitt-Engel ◽

Nadi Strohlein ◽

...

Keyword(s):

Functional Genomics ◽

Large Scale ◽

Reverse Genetics ◽

Expression Profiles ◽

Forward Genetics ◽

Large Set ◽

Knock Down ◽

Genome Wide ◽

Phenotypic Screen ◽

A Genome

Abstract Background Functional genomics uses unbiased systematic genome-wide gene disruption or analyzes natural variations such as gene expression profiles of different tissues from multicellular organisms to link gene functions to particular phenotypes. Functional genomics approaches are of particular importance to identify large sets of genes that are specifically important for a particular biological process beyond known candidate genes, or when the process has not been studied with genetic methods before. Results Here, we present a large set of genes whose disruption interferes with the function of the odoriferous defensive stink glands of the red flour beetle Tribolium castaneum. This gene set is the result of a large-scale systematic phenotypic screen using a reverse genetics strategy based on RNA interference applied in a genome-wide forward genetics manner. In this first-pass screen, 130 genes were identified, of which 69 genes could be confirmed to cause knock-down gland phenotypes, which vary from necrotic tissue and irregular reservoir size to irregular color or separation of the secreted gland compounds. The knock-down of 13 genes caused specifically a strong reduction of para-benzoquinones, suggesting a specific function in the synthesis of these toxic compounds. Only 14 of the 69 confirmed gland genes are differentially overexpressed in stink gland tissue and thus could have been detected in a transcriptome-based analysis. Moreover, of the 29 previously transcriptomics-identified genes causing a gland phenotype, only one gene was recognized by this phenotypic screen despite the fact that 13 of them were covered by the screen. Conclusion Our results indicate the importance of combining diverse and independent methodologies to identify genes necessary for the function of a certain biological tissue, as the different approaches do not deliver redundant results but rather complement each other. The presented phenotypic screen together with a transcriptomics approach are now providing a set of close to hundred genes important for odoriferous defensive stink gland physiology in beetles.

Download Full-text

Recent advances in functional genome analysis

F1000Research ◽

10.12688/f1000research.15274.1 ◽

2018 ◽

Vol 7 ◽

pp. 1968 ◽

Cited By ~ 3

Author(s):

Roderic Guigo ◽

Michiel de Hoon

Keyword(s):

Functional Genomics ◽

Human Genome ◽

Large Scale ◽

Massively Parallel Sequencing ◽

Genome Project ◽

Biological Traits ◽

Cellular Behavior ◽

Sequencing Technologies ◽

Recent Advances ◽

The Human Genome Project

At the beginning of this century, the Human Genome Project produced the first drafts of the human genome sequence. Following this, large-scale functional genomics studies were initiated to understand the molecular basis underlying the translation of the instructions encoded in the genome into the biological traits of organisms. Instrumental in the ensuing revolution in functional genomics were the rapid advances in massively parallel sequencing technologies as well as the development of a wide diversity of protocols that make use of these technologies to understand cellular behavior at the molecular level. Here, we review recent advances in functional genomic methods, discuss some of their current capabilities and limitations, and briefly sketch future directions within the field.

Download Full-text

RNAi-Based Functional Genomics Screening of the Human Kinome Identifies Major Growth Regulating Kinases in Acute Myeloid Leukemia Cells

Blood ◽

10.1182/blood.v112.11.2951.2951 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2951-2951

Author(s):

Raoul Tibes ◽

Ashish Choudhary ◽

Amanda Henrichs ◽

Sadia Guled ◽

Irma Monzon ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Acute Myeloid Leukemia ◽

Gene Silencing ◽

Functional Genomics ◽

Cell Lines ◽

Myeloid Leukemia ◽

Large Scale ◽

Cell Cycle Checkpoint ◽

Acute Myeloid

Abstract In order to improve treatment strategies for Acute Myeloid Leukemia (AML), we adapted a functional genomics approach using RNAi screening to identify molecular targets that are vital to the growth of AML. Herein we report the first large-scale kinome gene silencing screen in AML. A high throughput RNAi screen was developed for the efficient siRNA transfection of AML cell lines. Eight commercially available cationic lipid-based transfection reagents were tested for their ability to transfect several AML cell lines with siRNA. These extensive transfection optimization experiments identified two AML cells lines TF-1 and ML4 with up to 95–100 and 70–75% transfection efficiency respectively. Two independent replicate kinome screens were performed on both cell lines using a siRNA library targeting 572 kinase genes with 2 siRNA/gene. At 96 hours post transfection, cell proliferation was assessed and the B-score method was used to background correct and analyze the screening data. Several siRNA to specific kinases were identified that significantly inhibit cell proliferation of up to ~40–88%. Hits were defined at two thresholds: siRNA having a B-score of <−2 providing a statistically significance of p<0.05 (confidence of > 95%) and a cutoff B-score of <−1.5 providing greater than 87% confidence for each siRNA hit. Two different kinases (2 siRNA/gene/screen) were identified as major growth regulating kinases in TF1 cells with all 4 siRNA/gene having a B-score <−2. For two additional kinases, 3/4 siRNA for each gene had a Bscore <−2. Expanding the cutoff to a B-score <−1.5 three further kinases were targeted by at least 3/4 siRNA/gene. Similar analysis using the same criteria for ML4 cells identified one kinase targeted by 3/4 siRNA at a B-score <−2, seven kinases with 2/4 siRNA <−2 and two kinases with 3/4 siRNA/gene at a B-score of <−1.5. Common hits for both cell lines with at least 6/8 siRNA per gene from 4 screens performing at a B-score <−2 identified two kinases, one of them PLK1. Applying a B-score threshold of <−1.5, we identified five kinases for which at least 5/8 siRNA/gene from 4 screens met these criteria. Kinases/genes will be presented at the meeting.Confirmation of gene silencing and validation of growth response is currently underway for a subset of genes. Among the strongest hits are siRNA targeting PLK1, as well as siRNA targeting three other kinase-genes involved in regulating cell cycle progression and checkpoints and gene ontology (GO) analysis showed enrichment in cell cycle and cell cycle-checkpoint processes. Inhibitors against PLK1 and other kinase hits identified in the screen are in (pre)-clinical development and if confirmed, our experiments provide a strong rational to test these in AML. The application of RNAi based screening is useful in the identification of genes important in AML proliferation, which could serve as targets for therapeutic intervention and guide AML drug development. Furthermore, results from these types of functional genomics approaches hold promise to be rapidly translated into clinical application.

Download Full-text