Name: manipulation of gene function in mexican cavefish
Uploaded: 2019-04-22T13:00:00
Description: Watch this Scientific Journal Video about Manipulation of Gene Function in Mexican Cavefish at JoVE.com

Forfatterne takker Sunishka Thakur for henne hjelp i genotyperingteknologi og tenkelig oca2 mutant fisken avbildet i figur 2. Dette arbeidet ble støttet av National Science Foundation (NSF) prisen 1656574 til A.C.K., NSF award 1754321 JK og A.C.K., og National Institutes of Health (NIH) award R21NS105071 A.C.K. og E.R.D.

1. Morpholino oligo design
Merk: Sekvenser for A. mexicanus er tilgjengelig gjennom nasjonale senter for bioteknologi informasjon (NCBI) Gene og NCBI SRA (https://www.ncbi.nlm.nih.gov), samt fra Ensembl genomet leseren (https://www.ensembl.org). Når du utformer en morpholino for bruk i både overflaten og hulen boligen, er det avgjørende å identifisere alle genetisk variasjon mellom morphs på dette stadiet, slik disse genetisk områder kan unngås som mål for morpholinos. En polymo...

Her gitt vi en metode for å manipulere gen funksjon morpholinos, CRISPR/Cas9 gene redigering og transgenesis metodikk. Vell av genetisk teknologi og optimalisering av disse systemene i sebrafisk vil trolig tillate overføring av disse verktøyene i A. mexicanus med letthet52. Nyere funn har brukt disse metodene i A. mexicanus, men de forblir underutilized etterforskningen av ulike morfologiske, utviklingsmessige og atferdsmessige egenskaper i dette systemet30</...

Siden Darwins Origin of Species1fått forskere betydelig innsikt i hvordan trekk er formet evolusjonært svar på definerte miljøeffekter og økologiske press, takket være hule organismer2. Den meksikanske tetra, A. mexicanus, består av eyed forfedrenes 'overflate' populasjoner som bor elver i Mexico og sørlige Texas og minst 29 geografisk isolert bestander av avledede hule morphs bor Sierra del Abra og andre deler av nordøst Mexico3. En rekke hule-tilknyttede egenskaper har blitt identifisert i A. mexicanus, inkludert endrede oksygenforbruk, depigmentering, tap av øyne, og endret fôring og beite atferd4,5,6, 7,8,9. A. mexicanus presenterer en kraftig modell for å undersøke mekanismer for konvergent evolusjon en veldefinert evolusjonær historie, en detaljert karakterisering av økologiske miljøet og tilstedeværelsen av uavhengig utviklet seg hulen bestander10,11. Mange av de avledede hule egenskapene som finnes i cavefish, inkludert øye tap, sove tap, økt fôring, tap av skolegang, redusert aggresjon, og redusert stressresponser, har utviklet seg flere ganger gjennom uavhengige opprinnelse, ofte utnytte ulike genetisk veier mellom grotter8,12,13,14,15. Dette gjentas evolusjon er en kraftig del av A. mexicanus og kan gi innsikt i spørsmålet om hvordan genetiske systemer mer generelle kan være opprørt generere lignende fenotyper.
Mens anvendelsen av genetisk teknologi for mekanistisk etterforskningen av gen funksjon har blitt begrenset mange fiskearter (inkludert A. mexicanus), gir nylige fremskritt innen sebrafisk grunnlag for genetisk teknologiutvikling i fisk 16,17,18,19,20. Mange verktøy er mye brukt i sebrafisk for å manipulere genekspresjon, og gjennomføringen av disse prosedyrene har lenge blitt standardisert. For eksempel injeksjon av morpholino oligos (MOs) på encellede scenen selektivt blokkerer RNA og hindrer oversettelse for et kort timelige vindu under utvikling21,22. I tillegg gen-redigering tilnærminger, slik som clustered regelmessig interspaced kort palindromic gjentar (CRISPR) / CRISPR-assosiert protein 9 (Cas9) og transkripsjon aktivator som effektor nuclease (TALEN), tillater generering av definerte slettinger eller, i noen tilfeller, innsettinger gjennom en rekombinasjon i genomer19,20,23,24. Transgenesis brukes til å manipulere stabil genuttrykk eller funksjonen på en celle-type bestemt måte. Tol2 systemet brukes effektivt til å generere transgene dyr ved coinjecting transposase mRNA med en Tol2 DNA plasmider inneholder en transgene25,26. Tol2 systemet benytter Tol2 transposase av medaka å generere stabil germline innsettinger av transgene construct17. Genererer Tol2 transgenics innebærer coinjecting en plasmider inneholder en transgene flankert av Tol2 integrasjon områder og mRNA for Tol2 transposase17. Dette systemet har blitt brukt til å generere en rekke transgene linjer i sebrafisk og bruken har nylig utvidet til flere emergent modellsystemer, inkludert ciklider, killifish, Stingsild, og, mer nylig, den meksikanske cavefish27, 28,29,30.
Cavefish er et fascinerende biologiske system for Klargjørende mekanismer av egenskap evolusjon, har sin fulle kapasitet som en evolusjonær modell ikke blitt fullt utnyttet. Dette er delvis på grunn av en manglende evne til å manipulere genetiske og cellular fungere direkte31. Kandidat gener regulere komplekse egenskaper har blitt identifisert ved hjelp av kvantitative egenskap loci (QTL) studier, men validering av disse kandidat gener er vanskelig32,33,34. Nylig forbigående knockdown bruker morpholinos, gene redigering med CRISPR og TALEN, og bruk av Tol2-mediert transgenesis har blitt brukt til å undersøke genetisk grunnlag underliggende flere trekk35,36,37 ,38. Implementering og standardisering av disse teknikkene gjør manipulasjoner som forhøre molekylære og nevrale grunnlaget for biologiske egenskaper, inkludert manipulering av gen funksjon, merkingen av definerte celle populasjoner, og uttrykket av funksjonelle journalister. Mens den vellykkede implementeringen av disse genetiske verktøy for å manipulere genet eller cellulære funksjoner har blitt vist i emergent modellsystemer, er detaljert protokoller fortsatt mangler i A. mexicanus.
A. mexicanus gi viktig innsikt i mekanismer for utviklingen som svar på en endring miljøet og presentere mulighet til å identifisere romanen gener som regulerer ulike egenskaper. En rekke faktorer tyder på at A. mexicanus er en ekstremt tett modell for søker etablerte genomisk verktøy tilgjengelig i etablerte genetisk modeller, inkluderer evnen å lett vedlikeholde fisk i laboratorier, stor Foreldreomsorg og yngelens størrelse, åpenhet, sekvensert genomet og definerte opptreden analyser39. Her beskriver vi en metode for bruk av morpholinos, transgenesis og gene redigere overflaten og hule bestander av A. mexicanus. Bredere anvendelse av disse verktøyene i A. mexicanus vil tillate en mekanistisk undersøkelse molekylære prosesser underliggende utviklingen av utviklingsmessige, fysiologiske og behavioral forskjellene mellom cavefish og overflate fisk.

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manipulation of gene function in mexican cavefish

Hulen dyr gir et spennende system for gransker evolusjonære mekanismer og genetisk baser underliggende endringer i mange komplekse egenskaper, inkludert øye degenerasjon, albinisme, sove tap, hyperphagia og sensorisk prosessering. Arter av cavefish fra hele verden vise en konvergent evolusjon av morfologiske og atferdsmessige egenskaper på grunn av delt miljøbelastningene mellom forskjellige hule systemene. Mangfoldig hule arter har blitt studert i laboratoriet innstillingen. Den meksikanske tetra, Astyanax mexicanus, med seende og blind skjemaer har unik innsikt i biologiske og molekylære prosesser underliggende utviklingen av komplekse trekk og er godt klar som en ny modell. Mens kandidat gener regulere utviklingen av ulike biologiske prosesser er blitt identifisert i A. mexicanus, er muligheten til å validere en rolle for enkelte gener begrenset. Programmet transgenesis og gen-redigering teknologi har potensialet å overvinne denne betydelig hinder og undersøke mekanismene bak utviklingen av komplekse trekk. Her beskriver vi en annen metode for å manipulere genuttrykk i A. mexicanus. Metoder omfatter bruk av morpholinos, Tol2 transgenesis, og gen-redigering systemer, vanligvis brukes i sebrafisk og andre fisk modeller, manipulere gen funksjon i A. mexicanus. Disse protokollene inneholder detaljerte beskrivelser av tidsbestemte avl prosedyrer, innsamling av befruktet egg, injeksjoner og valg av genmodifiserte dyr. Disse metodologiske tilnærmingsmåter vil tillate etterforskningen av genetiske og nevrale mekanismene bak utviklingen av forskjellige trekk i A. mexicanus.

Flere bestander av hule-bolig A. mexicanus viser redusert søvn og økt våkenhet/aktivitet i forhold til deres overflaten-bolig fra Art14. Hypocretin/orexin (HCRT) er en høyt konservert neuropeptide, som fungerer for å øke våkenhet, avvik i HCRT veien forårsake narkolepsi i mennesker og andre pattedyr47,48. Vi har tidligere vist at hule A. mexicanus økt uttrykk for HCRT peptid, antyder at det økt uttrykket for den...

Watch this Scientific Journal Video about Manipulation of Gene Function in Mexican Cavefish at JoVE.com

Manipulation of Gene Function in Mexican Cavefish

Vi beskriver metoder for manipulering av gener i den evolusjonære modellsystem Astyanax mexicanus. Tre ulike teknikker er beskrevet: Tol2-mediert transgenesis, målrettet manipulering av genomet bruker CRISPR/Cas9 og knockdown uttrykk ved hjelp av morpholinos. Disse verktøyene skal lette direkte etterforskning av gener underliggende variasjon mellom overflaten og hulen boligen skjemaer.

Manipulering av gen funksjon i meksikansk Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

Huledyr gir et overbevisende system for å undersøke ulike morfologiske, utviklingsmessige og atferdsmessige egenskaper. Men en av de begrensende faktorene for å etablere dette modellsystemet er mangel på genetiske verktøy som ville tillate oss å manipulere genfunksjonen. Her demonstrerer vi tre forskjellige tilnærminger for å manipulere genfunksjonen i den meksikanske hulefisken Astyanax mexicanus.Disse verktøyene vil muliggjøre utredning av genene som ligger til grunn for naturlige variasjoner mellom overflatefisk og hulefisk og hvordan de forholder seg til fenotyper. Bethany Stahl, en talentfull postdoktor i laboratoriet mitt, vil demonstrere denne prosedyren. Før du begynner prosedyren, fyll en 100 milliliter petriskål med varm 3% agarose oppløst i fiskesystemvann og legg forsiktig en egginjeksjonsform i agarose i en 45 graders vinkel, før du sakte senker formen i agarose for å unngå å fange luft under formen.Når agarose har størknet, fjern forsiktig formen og oppbevar platen ved fire grader Celsius i opptil en uke. Trekk deretter nåler fra borosilikatglasskapillærer og en elektrodenåletrekker i henhold til produsentens retningslinjer. På injeksjonsdagen bruker du en glasspipette til å overføre encelledegg til injeksjonsplaten, og fylle opptil fem rader av injeksjonsplaten med 30 til 40 egg per rad.Hold eggene hydrert med en liten mengde fiskesystemvann. Tin Morpholino på is og klargjør injeksjonsoppløsningen slik at 400 picograms Morpholino injiseres per egg ved hjelp av en lang Mikrolaster pipettespiss eller legger til en to til fire mikroliter bolus til enden. Når alle nålene er lastet, bruk tang til å trimme den overskytende lengden fra injeksjonspinnespissene og monter den første nålen i en mikromanipulator som er koblet til en picoliter mikroinjektor.Deretter plasserer du injeksjonsfatet under et dissekeringsmikroskop og bruker mikromanipulatoren til å trenge inn i hvert egg med nålen før du injiserer en nanoliter morpholino injeksjonsoppløsning direkte inn i eggeplommen. For CRISPR injeksjon, bland 25 picograms av guide RNA med 300 picograms av CRISPR-assosiert protein 9 messenger RNA og injisere to nanoliter av den resulterende RNA-løsningen i hvert embryo som vist. For Tol2 transposase og Tol2 flankert plasmid injeksjon, kombinere 25 nanogram per mikroliter av Tol2 messenger RNA med 25 nanograms per mikroliter av Tol2 plasmid konstruere og fenol rød i RNase-fritt vann.Deretter injiserer du en nanoliter oppløsning i hvert embryo som nettopp demonstrert. For å screene de Morpholino-injiserte dyrene på fire dager etter injeksjon, avhengig av interessens gen, visualiser fiskelarver under et stereomikroskop for å observere fenotypen til de injiserte dyrene eller videoopptaket for å måle atferd. For å screene for CRISPR-indels, overfør CRISPR-injiserte embryoer til PCR-rør og trekk ut DNA i henhold til standard DNA-ekstraksjonsprotokoller for polymerasekjedereaksjon eller PCR-analyse.For å vurdere for mutagenese, kjør fem mikroliter av PCR-produktet på en 3% agarose gel på 70 volt i tre timer. Wild-type, ikke-mutagenisert DNA vil resultere i et distinkt bånd, mens mutant DNA vil resultere i et smørebånd. Hule En mexicanus injisert med en kontroll Morpholino viser betydelig mer lokomotorisk aktivitet og redusert søvn over en 24-timers periode, sammenlignet med overflatefisk injisert med en kryptert Morpholino.Injeksjonen av hypocretin orexin Morpholino har liten effekt på søvn i overflatefisk sammenlignet med kontrollinjisert fisk. I motsetning, hypocretin orexin knockdown via Morpholino injeksjon har en betydelig effekt på søvn i hule-bolig fisk, gir en direkte sammenheng mellom hypocretin orexin uttrykk og søvntap. CRPSR-mediert mutagenese av albinismegenet OCA2 i overflatefisk resulterer i noen individer homozygous for vill-type OCA2 positiv allele, og er forbundet med å huse pigmentering, mens personer som har to kopier av CRISPR-mutert OCA2 negativ allele er albino, gir en direkte kobling mellom mutant OCA2 genet og albinisme i hulefisk.Velg F0s positive for tol2-overført transgene til tilbake kryss til villtypen for å generere stabile F1-linjer. Dette gjør det mulig å leve kalsiumavbildning for å avdekke forskjeller i nevronal aktivitet, megling atferdsendringer i hulemiljøet, legge grunnlaget for uttrykk for mange ekstra transgener for å karakterisere og manipulere genfunksjon i A mexicanus. Det viktigste å huske er å laste eggene tett på platen, å trimme nålen riktig, og for å optimalisere mikroinjeksjonen.Etter en vellykket genetisk knockdown, transgene integrasjon, eller CRISPR-mediert genredigering, kan disse fiskene brukes til utviklings-, atferds- og hjerneaktivitetsanalyser.

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Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Genetisk manipulering av Plant Patogen<em&gt; Ustilago maydis</em&gt; Å studere Fungal Biology and Plant mikrobe interaksjoner

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Parallell Måling av biologiske klokke genekspresjon og hormon i Human primære cellekulturer

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Oppdrett og Double-stranded RNA-mediert Gene knockdown i Hide Beetle,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

En Standard metode for å undersøke stedet genetisk virkning som en funksjon av punkt mutasjon reparasjon katalysert av CRISPR/Cas9 og SsODN i menneskeceller

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

Generering av en gen-forstyrret Streptococcus mutans belastning uten Gene kloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

Manipulering av Ploidy i Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Bruke musen Oocytes å vurdere menneskelige gen funksjon under meiose jeg

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Culturing and Manipulation of O9-1 Neural Crest Cells

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.

O9-1 is a multipotent mouse neural crest cell line. Here we describe detailed step-by-step protocols for culturing O9-1 cells, differentiating O9-1 cells into specific cell types, and genetically manipulating O9-1 cells by using siRNA-mediated knockdown or CRISPR-Cas9 genome editing.

Dyrking og manipulering av O9-1 Neural Crest celler

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and ...

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

Heve den meksikanske Tetra Astyanax mexicanus for analyse av Post-larval fenotyper og hele-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

In vivo anvendelsen av fjernkontroll systemet for manipulering av endogene genuttrykk

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...