Understanding Of Gene Function Research Articles

Visualization Technique for Mutation Functional Analysis

A great body of research have been devoted in the past two decades to understanding gene functions, gene mutations, and mutation-disease relationships. In this paper, we present a method for exploring and understanding mutation functions using visualization and graphics representation. Functional annotation of human gene mutations is an important step in mutation pathogenicity prediction and understanding diseases progress and mechanisms. The presented visualization method is based on identifying the most significant and most specific sets of functions for a given set of mutations under a target disease or medical condition. The presented visualization allows for easy and effective understanding of the details and differences among the various sets of functions of the given mutations under certain disease or medical condition.

The History of Transgenesis.

A transgenic mouse carries within its genome an artificial DNA construct (transgene) that is deliberately introduced by an experimentalist. These animals are widely used to understand gene function and protein function. When addressing the history of transgenic mouse technology, it is apparent that a number of basic science research areas laid the groundwork for success. These include reproductive science, genetics and molecular biology, and micromanipulation and microscopy equipment. From reproductive physiology came applications on how to optimize mouse breeding, how to superovulate mice to produce zygotes for DNA microinjection or preimplantation embryos for combination with embryonic stem (ES) cells, and how to return zygotes and embryos to a pseudopregnant surrogate dam for gestation and birth. From developmental biology, it was learned how to micromanipulate embryos for morula aggregation and blastocyst microinjection and how to establish germline competent ES cells. From genetics came the foundational principles governing the inheritance of genes, the interactions of gene products, and an understanding of the phenotypic consequences of genetic mutations. From molecular biology came a panoply of tools and reagents that are used to clone DNA transgenes, to detect the presence of transgenes, to assess gene expression by measuring transcription, and to detect proteins in cells and tissues. Technical advances in light microscopes, micromanipulators, micropipette pullers, and ancillary equipment made it possible for experimentalists to insert thin glass needles into zygotes or embryos under controlled conditions to inject DNA solutions or ES cells. To fully discuss the breadth of contributions of these numerous scientific disciplines to a comprehensive history of transgenic science is beyond the scope of this work. Examples will be used to illustrate scientific developments central to the foundation of transgenic technology and that are in use today.

A CRISPR/Cas9 method to generate heterozygous alleles in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a genetically facile organism, yet multiple CRISPR/Cas9 techniques are widely used to edit its genome more efficiently and cost effectively than conventional methods. The absence of selective markers makes CRISPR/Cas9 editing particularly useful when making mutations within genes or regulatory sequences. Heterozygous mutations within genes frequently arise in the winners of evolution experiments. The genetic dissection of heterozygous alleles can be important to understanding gene structure and function. Unfortunately, the high efficiency of genome cutting and repair makes the introduction of heterozygous alleles by standard CRISPR/Cas9 technique impossible. To be able to quickly and reliably determine the individual phenotypes of the thousands of heterozygous mutations that can occur during directed evolutions is of particular interest to industrial strain improvement research. In this report, we describe a CRISPR/Cas9 method that introduces specific heterozygous mutations into the S.cerevisiae genome. This method relies upon creating silent point mutations in the protospacer adjacent motif site or removing the protospacer adjacent motif site entirely to stop the multiple rounds of genome editing that prevent heterozygous alleles from being generated. This technique should be able to create heterozygous alleles in other diploid yeasts and different allelic copy numbers in polyploid cells.

Comparison between Lipofectamine RNAiMAX and GenMute transfection agents in two cellular models of human hepatoma.

RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-trasfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR finding similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have a higher viability with respect to Lipofectamine RNAiMAXadministered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.

First person – Francesco Chiani and Tiziana Orsini

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms (DMM), helping early-career researchers promote themselves alongside their papers. Francesco Chiani and Tiziana Orsini are co-first authors on ‘Functional loss of Ccdc151 leads to hydrocephalus in a mouse model of primary ciliary dyskinesia’, published in DMM. Francesco is a researcher (PhD) in the lab of Fabio Mammano (Italian IMPC/INFRANFRONTIER delegate) at the Institute of Biochemistry and Cell Biology – Italian National Research Council, Monterotondo, Rome, Italy, where he is interested in engineering and studying novel mouse models for human disease with the aim to understand gene function in normal physiology and pathological conditions. Tiziana is a CNR Technologist in the lab of Fabio Mammano, studying the function of genes in normal conditions and during disease development in mouse models by applying the micro-computed tomography (micro-CT) imaging technique.

ISuRe-Cre is a genetic tool to reliably induce and report Cre-dependent genetic modifications

Most biomedical research aimed at understanding gene function uses the Cre-Lox system, which consists of the Cre recombinase-dependent deletion of genes containing LoxP sites. This system enables conditional genetic modifications because the expression and activity of the recombinase Cre/CreERT2 can be regulated in space by tissue-specific promoters and in time by the ligand tamoxifen. Since the precise Cre-Lox recombination event is invisible, methods were developed to report Cre activity and are widely used. However, numerous studies have shown that expression of a given Cre activity reporter cannot be assumed to indicate deletion of other LoxP-flanked genes of interest. Here, we report the generation of an inducible dual reporter-Cre mouse allele, iSuRe-Cre. By significantly increasing Cre activity in reporter-expressing cells, iSuRe-Cre provides certainty that these cells have completely recombined floxed alleles. This genetic tool increases the ease, efficiency, and reliability of conditional mutagenesis and gene function analysis.

Gcorn Plant: A Database for Retrieving Functional and Evolutionary Traits of Plant Genes.

Gene homology helps us understand gene function and speciation. The number of plant genes and species registered in public databanks is continuously increasing. It is useful to associate homologous genes of various plants to better understand plant speciation. We designed the Gcorn plant database for the retrieval of information on homology and evolution of a plant gene of interest. Amino acid sequences of 73 species (62 land plants and 11 green algae), containing 2,682,261 sequences, were obtained from the National Center for Biotechnology Information (NCBI) Reference Sequence database. Based on NCBI BLAST searches between these sequences, homologous genes were grouped at various thresholds of homology indices devised by the authors. To show functional and evolutionary traits of a gene of interest, a phylogenetic tree, connecting genes with high homology indices, and line charts of the numbers of genes with various homology indices, are depicted. In addition, such indices are projected on a network graph in which species studied are connected based on the ratios of homologous genes, and on a phylogenetic tree for species based on NCBI Taxonomy. Gcorn plant provides information on homologous genes at various virtual time points along with speciation in plants.

Twenty-Seven Tamoxifen-Inducible iCre-Driver Mouse Strains for Eye and Brain, Including Seventeen Carrying a New Inducible-First Constitutive-Ready Allele.

To understand gene function, the cre/loxP conditional system is the most powerful available for temporal and spatial control of expression in mouse. However, the research community requires more cre recombinase expressing transgenic mouse strains (cre-drivers) that restrict expression to specific cell types. To address these problems, a high-throughput method for large-scale production that produces high-quality results is necessary. Further, endogenous promoters need to be chosen that drive cell type specific expression, or we need to further focus the expression by manipulating the promoter. Here we test the suitability of using knock-ins at the docking site 5′ of Hprt for rapid development of numerous cre-driver strains focused on expression in adulthood, using an improved cre tamoxifen inducible allele (icre/ERT2), and testing a novel inducible-first, constitutive-ready allele (icre/f3/ERT2/f3). In addition, we test two types of promoters either to capture an endogenous expression pattern (MaxiPromoters), or to restrict expression further using minimal promoter element(s) designed for expression in restricted cell types (MiniPromoters). We provide new cre-driver mouse strains with applicability for brain and eye research. In addition, we demonstrate the feasibility and applicability of using the locus 5′ of Hprt for the rapid generation of substantial numbers of cre-driver strains. We also provide a new inducible-first constitutive-ready allele to further speed cre-driver generation. Finally, all these strains are available to the research community through The Jackson Laboratory.

Streamlined computational pipeline for genetic background characterization of genetically engineered mice based on next generation sequencing data

BackgroundGenetically engineered mice (GEM) are essential tools for understanding gene function and disease modeling. Historically, gene targeting was first done in embryonic stem cells (ESCs) derived from the 129 family of inbred strains, leading to a mixed background or congenic mice when crossed with C57BL/6 mice. Depending on the number of backcrosses and breeding strategies, genomic segments from 129-derived ESCs can be introgressed into the C57BL/6 genome, establishing a unique genetic makeup that needs characterization in order to obtain valid conclusions from experiments using GEM lines. Currently, SNP genotyping is used to detect the extent of 129-derived ESC genome introgression into C57BL/6 recipients; however, it fails to detect novel/rare variants.ResultsHere, we present a computational pipeline implemented in the Galaxy platform and in BASH/R script to determine genetic introgression of GEM using next generation sequencing data (NGS), such as whole genome sequencing (WGS), whole exome sequencing (WES) and RNA-Seq. The pipeline includes strategies to uncover variants linked to a targeted locus, genome-wide variant visualization, and the identification of potential modifier genes. Although these methods apply to congenic mice, they can also be used to describe variants fixed by genetic drift. As a proof of principle, we analyzed publicly available RNA-Seq data from five congenic knockout (KO) lines and our own RNA-Seq data from the Sall2 KO line. Additionally, we performed target validation using several genetics approaches.ConclusionsWe revealed the impact of the 129-derived ESC genome introgression on gene expression, predicted potential modifier genes, and identified potential phenotypic interference in KO lines. Our results demonstrate that our new approach is an effective method to determine genetic introgression of GEM.

Genetically engineered stem cell‐derived neurons can be rendered resistant to alpha‐synuclein aggregate pathology

Genetically engineered stem cell‐derived neurons can be rendered resistant to alpha‐synuclein aggregate pathology

Inducible IFN-γ Expression for MHC-I Upregulation in Devil Facial Tumor Cells.

The Tasmanian devil facial tumor (DFT) disease has led to an 80% reduction in the wild Tasmanian devil (Sarcophilus harrisii) population since 1996. The limited genetic diversity of wild devils and the lack of MHC-I expression on DFT cells have been implicated in the lack of immunity against the original DFT clonal cell line (DFT1). Recently, a second transmissible tumor of independent origin (DFT2) was discovered. Surprisingly, DFT2 cells do express MHC-I, but DFT2 cells appear to be on a trajectory for reduced MHC-I expression in vivo. Thus, much of the ongoing vaccine-development efforts and conservation plans have focused on MHC-I. A major limitation in conservation efforts is the lack of species-specific tools to understand Tasmanian devil gene function and immunology. To help fill this gap, we developed an all-in-one Tet-Off vector system to regulate expression of IFN-γ in DFT cells (DFT1.Tet/IFN-γ). IFN-γ can have negative effects on cell proliferation and viability; thus, doxycycline was used to suppress IFN-γ production whilst DFT1.Tet/IFN-γ cells were expanded in cell culture. Induction of IFN-γ following removal of doxycycline led to upregulation of MHC-I but also the inhibitory checkpoint molecule PD-L1. Additionally, DFT1.Tet/IFN-γ cells were capable of stimulating MHC-I upregulation on bystander wild type DFT cells in co-culture assays in vitro. This system represents a major step forward in DFT disease immunotherapy and vaccine development efforts, and ability to understand gene function in devils. Importantly, the techniques are readily transferable for testing gene function in DFT2 cells and other non-traditional species.

Ranking Cancer Proteins by Integrating PPI Network and Protein Expression Profiles.

Proteomics, the large-scale analysis of proteins, is contributing greatly to understanding gene function in the postgenomic era. However, disease protein ranking using shotgun proteomics data has not been fully evaluated. In this study, we prioritized disease-related proteins by integrating the protein-protein interaction (PPI) network and protein differential expression profiles from colon and rectal cancer (CRC) or breast cancer (BC) proteomics. We applied Local Ranking (LR) and Global Ranking (GR) methods in network with three kinds of protein sets as a priori knowledge, which were known disease proteins (KDPs) that were collected from the Online Mendelian Inheritance in Man (OMIM) database, differentially expressed proteins (DEPs), and the collection of KDPs and their direct neighborhood with differential expression (eKDPs). The cross-validations showed that GR method outperformed LR method while using eKDPs as the initial training showed significantly higher accuracy compared to using the other two a priori sets. And then we validated the top ranked proteins using RNAi-based loss-of-function screens in the DepMap database. The results showed that 75% of top 20 proteins in CRC are necessary for tumor survival. In summary, the network-based Global Ranking with protein differential expression can efficiently prioritize cancer-related proteins and discover new candidate cancer genes or proteins.

Efficient Gene Deletion Method for Listeria monocytogenes.

Inactivation or deletion of genes allows for investigation and understanding of gene function. To facilitate markerless gene deletion in Listeria monocytogenes, we developed a new suicide plasmid (pHoss1). pHoss1 contains the pMAD backbone, the secY antisense cassette from pIMAY driven by an inducible Pxyl/tetO promoter, a heat-sensitive origin of replication, four unique restriction sites (SalI, EcoRI, SmaI, and NcoI), and erythromycin resistance gene. We demonstrated that pHoss1 is very efficient for introducing mutations into different L. monocytogenes strains. In this chapter, we include a brief description of pHoss1 and the method used for gene deletion in L. monocytogenes using pHoss1.

Designing siRNA and Evaluating Its Effect on RNA Targets Using qPCR and Western Blot.

The discovery of the RNA interference (RNAi) pathway followed by the usage of synthetic short-interfering RNAs (siRNA) has contributed greatly to the understanding of gene function. Carefully designed siRNAs can considerably improve siRNA specificity leading to more accurate and efficient gene silencing. Evaluation of gene knockdown is vital for optimization of siRNA efficacy. Here we describe the fundamental principles of siRNA design and strategies for evaluating gene knockdown.

ISOdb: A Comprehensive Database of Full-Length Isoforms Generated by Iso-Seq.

The accurate landscape of transcript isoforms plays an important role in the understanding of gene function and gene regulation. However, building complete transcripts is very challenging for short reads generated using next-generation sequencing. Fortunately, isoform sequencing (Iso-Seq) using single-molecule sequencing technologies, such as PacBio SMRT, provides long reads spanning entire transcript isoforms which do not require assembly. Therefore, we have developed ISOdb, a comprehensive resource database for hosting and carrying out an in-depth analysis of Iso-Seq datasets and visualising the full-length transcript isoforms. The current version of ISOdb has collected 93 publicly available Iso-Seq samples from eight species and presents the samples in two levels: (1) sample level, including metainformation, long read distribution, isoform numbers, and alternative splicing (AS) events of each sample; (2) gene level, including the total isoforms, novel isoform number, novel AS number, and isoform visualisation of each gene. In addition, ISOdb provides a user interface in the website for uploading sample information to facilitate the collection and analysis of researchers' datasets. Currently, ISOdb is the first repository that offers comprehensive resources and convenient public access for hosting, analysing, and visualising Iso-Seq data, which is freely available.

CRISPR/Cas9 for Mutagenesis in Rice.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) provides a workhorse for genome editing biotechnology. CRISPR/Cas9 tailored for enabling genome editing has been extensively interrogated and widely utilized for precise genomic alterations in eukaryotic organisms including in plant species. The technology holds the great promise to better understand gene functions, elucidate networks, and improve the performance of crop plants such as increasing grain yields, improving nutritional content, and better combating the biotic and abiotic stresses. Various methods or protocols specific for different plant species have been established. Here, we present a CRISPR/Cas9-mediated genome editing protocol in rice, including detailed information about single-guide RNA design, vector construction, plant transformation, and mutant screening processes.

Agrobacterium tumefaciens-Mediated Transformation of Tomato.

Tomato is both an important food crop and serves as a model plant species that is used for various research investigations including understanding gene function. Transformation is commonly utilized to facilitate these investigations in combination with all the extensive genetic and genomic resources available for tomato. The transformation protocol routinely used in our laboratory has been applied to many different tomato genotypes and relies on Agrobacterium tumefaciens infection of young cotyledon sections. We have used vector systems for overexpression, RNA interference for gene silencing, and CRISPR/Cas9 for genome editing. Vectors used to design gene constructs contained selectable marker genes that conferred resistance to kanamycin, hygromycin, and the herbicide component, bialaphos. The protocol we follow for Agrobacterium-mediated transformation of both cultivated and wild species of tomato is detailed in this chapter.

An Efficient Genome Editing Strategy To Generate Putative Null Mutants in Caenorhabditis elegans Using CRISPR/Cas9.

Null mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long knock-in cassette (STOP-IN) into the early exons of target genes. This STOP-IN cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two ∼35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2. We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting the exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for 20 additional genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.

Measuring Food Anticipation in Mice.

The interplay between the circadian system and metabolism may give animals an evolutionary advantage by allowing them to anticipate food availability at specific times of the day. Physiological adaptation to feeding time allows investigation of animal parameters and comparison of food anticipation between groups of animals with genetic alterations and/or post pharmacological intervention. Such an approach is vital for understanding gene function and mechanisms underlying the temporal patterns of both food anticipation and feeding. Exploring these mechanisms will allow better understanding of metabolic disorders and might reveal potential new targets for pharmacological intervention. Changes that can be easily monitored and that represent food anticipation on the level of the whole organism are a temporarily restricted increase of activity and internal body temperature.

The golden (Syrian) hamster as a model for the study of reproductive biology: Past, present, and future.

BackgroundThe golden (Syrian) hamster (Mesocricetus auratus) is a small rodent that belongs to the Cricetidae family. It has several unique features that are advantageous for the study of reproductive and developmental biology, including a consistent estrous cycle (4 days), high responsiveness to conventional superovulation regimens, and the short gestation period (16 days).MethodsBased on the published reports, the development in assisted reproductive technology (ART) in the golden hamsters was summarized.Main findingsThe technical ease of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in this species has contributed to our understanding of the basic mechanisms of mammalian fertilization in the last century. However, a strong developmental block in vitro of hamster embryos and unavailability of gene‐modified strains has hampered its broader use in biomedical fields. A recently developed in vivo transfection method has enabled us to generate gene knockout hamsters without any major obstacles. It would be interesting to revisit the genes whose functions could not be identified using mouse models.ConclusionThe authors expect that gene knockout hamsters might be able to substitute for mice—at least in part—for better understanding of gene functions in mammals including humans.