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 takke Sunishka Thakur for hendes hjælp i genotypebestemmelse og imaging oca2 mutant fisk afbildet i figur 2. Dette arbejde blev støttet af National Institutes of Health (NIH) award R21NS105071 til A.C.K. og E.R.D., National Science Foundation (NSF) award 1656574 til A.C.K. og NSF award 1754321 J.K. og A.C.K.

1. Morpholino oligo design
Bemærk: Sekvenser for A. mexicanus er tilgængelige via nationale Center for bioteknologi Information (NCBI) genet og NCBI SRA (https://www.ncbi.nlm.nih.gov), samt fra Ensembl genom browser (https://www.ensembl.org). Når du udformer en morpholino til brug i både overflade - og hulen-boligen former, er det afgørende at identificere eventuelle genetiske variation mellem morphs i denne fase, så disse genetiske regioner kan undgås som mål for morpholinos. E...

Her, leverer vi en metode for at manipulere genfunktion ved hjælp af morpholinos, CRISPR/Cas9 gen redigering og transgenese metode. Rigdommen af genteknologi og optimering af disse systemer i zebrafisk vil sandsynligvis give mulighed for overførsel af disse værktøjer til A. mexicanus med lethed52. De seneste resultater har brugt disse metoder i A. mexicanus, men de forbliver underudnyttede i undersøgelsen af forskellige morfologiske, udviklingsmæssige og adfærdsmæssige tr...

Siden Darwins Arternes oprindelse1, har forskere fået dybtgående indsigt i hvordan træk er formet evolutionært som svar på definerede miljømæssigt og økologisk pres, takket være hule organismer2. Den mexicanske tetra, A. mexicanus, består af eyed forfædres 'overflade' befolkning, der bebor floder i hele Mexico og det sydlige Texas og mindst 29 geografisk isolerede befolkninger af afledte cave morphs bebo Sierra del Abra og andre områder af nordøstlige Mexico3. En række af grotte-associerede træk er blevet identificeret i A. mexicanus, herunder ændret iltforbrug, depigmentering, tab af øjne, og ændret fodring og fouragering adfærd4,5,6, 7,8,9. A. mexicanus præsenterer en kraftfuld model for at undersøge mekanismerne på konvergent evolution på grund af en veldefineret evolutionære historie, en detaljeret beskrivelse af økologiske miljø og tilstedeværelsen af selvstændigt udviklet cave populationer10,11. Mange af de hule-afledte træk, der er til stede i cavefish, herunder øjet tab, sove tab, øget fodring, tab af skolegang, reduceret aggression, og reduceret stress svar, har udviklet sig flere gange gennem uafhængige oprindelse, ofte udnytter forskellige genetiske veje mellem caves8,12,13,14,15. Dette gentages evolution er en kraftfuld aspekt af A. mexicanus system og kan give indsigt i den mere generelle spørgsmål om hvordan genetiske systemer kan være rystet til at generere tilsvarende fænotyper.
Mens anvendelsen af genteknologi for mekanistisk undersøgelse af genfunktioner har været begrænset i mange fiskearter (herunder A. mexicanus), danne de seneste fremskridt i zebrafisk grundlag for genetisk teknologiudvikling i fisk 16,17,18,19,20. Adskillige værktøjer er meget udbredt i zebrafisk for at manipulere genekspression, og gennemførelsen af disse procedurer har længe blevet standardiseret. For eksempel, injektion af morpholino oligos (MOs) på stadiet encellede selektivt blokerer RNA og forhindrer oversættelse til et korte tidsmæssige vindue under udvikling21,22. Desuden gen-redigering metoder, såsom grupperet regelmæssigt interspaced korte palindromiske gentagelser (CRISPR) / CRISPR-forbundet protein 9 (Cas9) og transkription aktivere-lignende effektor nukleasen (TALEN), giver mulighed for generation af definerede sletninger eller i nogle tilfælde, indsættelser gennem en rekombination i genomer19,20,23,24. Transgenese bruges til at manipulere stabil genekspression eller funktion i en celletype specifikke måde. Tol2 systemet er brugt effektivt til at generere transgene dyr af coinjecting transposase mRNA med en Tol2 DNA plasmid indeholder et transgen25,26. Tol2 system udnytter Tol2 transposase af medaka til at generere stabile kønscelleoverførsel indsætninger af transgene construct17. Generere Tol2 transgenics indebærer coinjecting et plasmid som indeholder en transgen flankeret af Tol2 integrering websteder og mRNA for Tol2 transposase17. Dette system er blevet brugt til at generere et array af transgene linjer i zebrafisk og dets anvendelse har for nylig udvidet til yderligere emergent modelsystemer, herunder cichlider, killifish, Hundestejle, og mere for nylig, den mexicanske cavefish27, 28,29,30.
Mens cavefish er et fascinerende biologisk system for informative mekanismer af trait evolution, har sin fulde kapacitet som en evolutionær model ikke blevet fuldt udnyttet. Dette er delvis på grund af manglende evne til at manipulere genetiske og cellulære funktion direkte31. Kandidat gener regulering komplekse træk er blevet identificeret ved hjælp af kvantitative træk loci (QTL) undersøgelser, men valideringen af disse kandidat gener har været vanskelige32,33,34. For nylig, forbigående knockdown ved hjælp af morpholinos, gen redigering bruger CRISPR og TALEN, og brugen af Tol2-medieret transgenese har været brugt til at undersøge det genetiske grundlag bag en række træk35,36,37 ,38. Implementering og standardisering af disse teknikker vil give mulighed for manipulationer, at afhøre de molekylære og neurale fundament for biologiske egenskaber, herunder manipulation af genfunktioner, mærkning af definerede cellepopulationer, og udtryk for funktionelle journalister. Der henviser til, at en vellykket gennemførelse af disse genetiske værktøjer til at manipulere gen eller cellulære funktion er blevet påvist i emergent modelsystemer, er detaljerede protokoller stadig mangler i A. mexicanus.
A. mexicanus har kritisk indsigt i mekanismerne i evolution som reaktion på et skiftende miljø og præsentere mulighed for at identificere nye gener, der regulerer forskellige træk. En række faktorer tyder på, at A. mexicanus er en ekstremt tractable model for anvendelse af etablerede genomisk værktøjer i øjeblikket tilgængelig i etablerede genetiske modeller, herunder evnen til at let fastholde fisk i laboratorier, store kuld størrelse, gennemsigtighed, sekventeret genom og definerede adfærdsmæssige assays39. Her, beskriver vi en metode til anvendelse af morpholinos, transgenese og gen redigering i overfladen og cave populationer af A. mexicanus. Den bredere anvendelse af disse værktøjer i A. mexicanus muliggør en mekanistisk undersøgelse af de molekylære processer bag udviklingen af udviklingsmæssige, fysiologiske og adfærdsmæssige forskelle mellem cavefish og overflade fisk.

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manipulation of gene function in mexican cavefish

Cave dyr giver en overbevisende system for at undersøge den evolutionære mekanismer og genetiske grundlag bag ændringer i mange komplekse træk, herunder øjet degeneration, albinisme, søvn tab, Hyperfagi og sensoriske forarbejdning. Arter af cavefish fra hele verden vist en konvergent evolution morfologiske og adfærdsmæssige træk på grund af delte miljøbelastning mellem forskellige grotte systemer. Forskellige cave arter er blevet undersøgt i laboratoriet indstilling. Den mexicanske tetra, Astyanax mexicanus, med seende og blinde former, har givet unik indsigt i biologiske og molekylære processer bag udviklingen af komplekse træk og er godt klar som en spirende modelsystem. Mens kandidat gener regulering udviklingen af forskellige biologiske processer er blevet identificeret i A. mexicanus, har evnen til at validere en rolle for enkelte gener været begrænset. Anvendelsen af transgenese og gen-redigering teknologi har potentialet til at overvinde denne store forhindring og til at efterforske de mekanismer, der ligger til grund for udviklingen af komplekse træk. Her, beskriver vi en anden metode til at manipulere genekspression i A. mexicanus. Metoder omfatter brug af morpholinos, Tol2 transgenese, og gen-redigering systemer, er almindeligt anvendt i zebrafisk og andre fisk modeller, for at manipulere genfunktion i A. mexicanus. Disse protokoller indeholder detaljerede beskrivelser af timet avl procedurer, indsamling af befrugtede æg, injektioner og udvælgelse af genetisk modificerede dyr. Disse metodiske tilgange giver mulighed for undersøgelse af de genetiske og neurale mekanismer bag udviklingen af forskellige træk i A. mexicanus.

Flere bestande af hulen-boligen A. mexicanus Vis reduceret søvn og øget vågenhed/aktivitet i forhold til deres overflade-bolig artsfæller14. Hypocretin/orexin (HCRT) er en yderst velbevarede neuropeptid, som virker til at øge vågenhed, og afvigelser i HCRT pathway forårsager narkolepsi hos mennesker og andre pattedyr47,48. Vi har tidligere vist, at hulen A. mexicanus har øget udtryk for HCRT peptid, tyder på, at ...

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 tilgange til manipulering af gener i den evolutionære modelsystem Astyanax mexicanus. Tre forskellige teknikker er beskrevet: Tol2-medieret transgenese, målrettede manipulation af genomet ved hjælp af CRISPR/Cas9 og knockdown ytringsfriheden ved hjælp af morpholinos. Disse værktøjer bør lette den direkte undersøgelse af gener, der ligger til grund for variationen mellem overflade - og hulen-boligen former.

Manipulation af genfunktioner i mexicansk Cavefish

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

Cave dyr giver en overbevisende system til at undersøge forskellige morfologiske, udviklingsmæssige, og adfærdsmæssige træk. Men en af de begrænsende faktorer i etableringen af denne model system er en mangel på genetiske værktøjer, der ville give os mulighed for at manipulere genfunktion. Her viser vi tre forskellige tilgange til at manipulere genfunktion i den mexicanske hule fisk, Astyanax mexicanus.Disse værktøjer vil gøre det muligt at undersøge de gener, der ligger til grund for naturlige variationer mellem overfladefisk og hulefisk, og hvordan de relaterer til fænotyper. Bethany Stahl, en talentfuld postdoc i mit laboratorium vil demonstrere denne procedure. Før du begynder proceduren, fylde en 100 milliliter petriskål med varm 3% agarose opløst i fiskesystemet vand og omhyggeligt placere et æg sprøjteform i agarose i en 45 graders vinkel, før langsomt sænke formen ind i agarose for at undgå fældefangst luft under formen.Når agarose har størknet, forsigtigt fjerne formen derefter gemme pladen ved fire grader Celsius i op til en uge. Træk derefter nåle fra borosilikatglaskapillærer og en elektrode nål puller i henhold til producentens retningslinjer. På injektionsdagen skal du bruge en glaspipette til at overføre enkeltcelleæg i injektionspladen og fylde op til fem rækker af injektionspladen med 30 til 40 æg pr. række.Hold æggene hydreret med en lille mængde fisk system vand. Tø Morpholino op på is og klargør injektionsopløsningen, så 400 picogrammer af Morpholino injiceres pr. æg ved hjælp af en lang Microloader pipettespids eller tilsætning af en to til fire mikroliterbolus til enden. Når alle nåle er blevet indlæst, skal du bruge vicesrene til at trimme den overskydende længde fra injektionsnålenspidserne og montere den første nål i en mikromanipulator, der er forbundet med en picoliter microinjector.Derefter skal injektionsskålen sættes under et dissekerende mikroskop, og mikromanipulatoren anvendes til at trænge ind i hvert æg med nålen, før du injicerer en nanoliter af Morpholino-injektionsopløsningen direkte i æggeblomme. Til CRISPR-injektion blandes 25 picogrammer af guide RNA med 300 picogrammer af CRISPR-associeret protein 9 messenger RNA, og der injiceres to nanoliter af den resulterende RNA-opløsning i hvert foster som påvist. For Tol2 transposase og Tol2 flankeret plasmid injektion, kombinere 25 nanogram per mikroliter af Tol2 messenger RNA med 25 nanogram per mikroliter af Tol2 plasmid konstruere og phenol rød i RNase-frit vand.Injicer derefter en nanoliter opløsning i hvert foster som netop påvist. For at screene De Morpholino-injicerede dyr efter injektionen, afhængigt af det pågældende gen, visualisere fiskelarver under et stereomikroskop for at observere fænotypen af de injicerede dyr eller videooptagelsen for at måle adfærd. For at screene for CRISPR indels skal crispr-indsprøjtede embryoner overføres til PCR-rør og udtrække DNA'et i henhold til standard-DNA-ekstraktionsprotokoller for polymerasekædereaktion eller PCR-analyse.For at vurdere for mutagenese køres der fem mikroliter af PCR-produktet på en 3% agarosegel ved 70 volt i tre timer. Wild-type, ikke-mutagenized DNA vil resultere i en særskilt band, mens mutant DNA vil resultere i en smeary band. Cave En mexicanus injiceres med en kontrol Morpholino udviser betydeligt mere bevægelsesaktivitet og reduceret søvn over en 24-timers periode, sammenlignet med overfladefisk injiceret med en krypteret Morpholino.Injektionen af hypocretin ellerexin Morpholino har ringe effekt på søvn i overfladefisk sammenlignet med kontrolinjekterede fisk. I modsætning hertil, hypocretin orexin knockdown via Morpholino injektion har en betydelig effekt på søvn i hule-bolig fisk, hvilket giver en direkte sammenhæng mellem hypocretin orexin udtryk og søvntab. CRPSR-medieret mutagenesis af albinisme genet OCA2 i overflade fisk resulterer i nogle individer homozygot for den vilde type OCA2 positiv allel, og er forbundet med husly pigmentering, mens personer, der har to kopier af CRISPR-muterede OCA2 negative allel er albino, hvilket giver en direkte forbindelse mellem mutant OCA2 genet og albinisme i hule fisk.Vælg F0's positive for Tol2 transmitterede transgene til tilbage kryds til den vilde type for at generere stabile F1 linjer. Dette gør det muligt levende calcium billeddannelse for at afdække forskelle i neuronal aktivitet, mægle adfærdsmæssige ændringer i hulen miljø, om grundlaget for udtrykket af mange yderligere transgenes at karakterisere og manipulere genfunktion i A mexicanus. De vigtigste ting at huske er at indlæse æggene stramt på pladen, at trimme nålen ordentligt, og at optimere mikroinjektion.Efter en vellykket genetisk knockdown, transgene integration, eller CRISPR-medieret genredigering, kan disse fisk bruges til udviklingsmæssige, adfærdsmæssige, og hjerne aktivitet analyser.

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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 Manipulation af afgroedesygdomme<em&gt; Ustilago maydis</em&gt; At studere Fungal Biology og 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 ...

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.

Parallel Måling af døgnrytmen ur genekspression og hormon sekretion 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.

Opdræt og dobbelt-strenget RNA-medieret Gene Knockdown i Skjul 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 standardmetode til at undersøge ejendommen mutagenicitet som en funktion af punkt Mutation reparation katalyseret af CRISPR/Cas9 og SsODN i humane celler

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.

Generation af en gen-forstyrret Streptococcus mutans stamme uden 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.

Manipulation af Ploidi 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.

Ved hjælp af musen oocyter at vurdere menneskelige genfunktion 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.

Dyrkning og Manipulation af O9-1 neurale 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.

At hæve den mexicanske Tetra Astyanax mexicanus for analyse af Post-larval fænotyper og hele-mount Immunohistokemi

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 anvendelse af sen-kontrol-System for Manipulation af endogene genekspression

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