Ved hjælp af musen oocyter at vurdere menneskelige genfunktion under meiose jeg
Summary
Som de genetiske varianter forbundet med menneskelig sygdom begynder at blive afdækket, det stadig vigtigere at udvikle systemer, som hurtigt vurdere den biologiske betydning af de identificerede varianter. Denne protokol beskriver metoder til evaluering menneskeligt genfunktion under kvindelige meiose jeg bruger musen oocyter.
Abstract
Embryonale aneuploidi er den største genetiske årsag til infertilitet hos mennesker. De fleste af disse begivenheder stammer under kvindelige meiose, og omend positivt korreleret med moderens alder, alder alene er ikke altid intelligent af risikoen for at generere et aneuploid Foster. Derfor kan gen-varianter tegner sig for forkert kromosom segregation under oogenesen. Adgang til menneskelige oocyter er begrænset til forskningsformål, en række assays blev udviklet for at studere menneskelige genfunktion under meiose jeg bruger musen oocyter. Først, messenger RNA (mRNA) genet og genet variant af interesse er microinjected til prophase jeg-anholdt mus oocyter. Efter giver tid til udtryk, oocyter udgives synkront i meiotiske modning at fuldføre meiose jeg. Ved tagging mRNA med en sekvens af en fluorescerende reporter, såsom grøn fluorescerende proteiner (normal god landbrugspraksis), kan lokalisering af det menneskelige protein vurderes ud over de fænotypiske forandringer. For eksempel, vinde eller tab af funktion kan undersøges ved at etablere forsøgsbetingelser, der udfordrer gen produktet til fix meiotiske fejl. Selv om dette system er en fordel i at undersøge menneskelige protein funktion under oogenesen, bør passende fortolkning af resultater foretages, da protein udtryk ikke er på endogene niveauer og, medmindre kontrolleret for (dvs. bankede ud eller ned) er murine homologs også til stede i systemet.
Introduction
Barnløshed er en sygdom, der rammer 10-15% af den menneskelige befolkning af fødedygtige alder 1, hvorfra næsten halvdelen søge lægebehandling 2. Selv om årsagen til barnløshed er mangfoldigt og i mange tilfælde multifaktoriel, er den mest almindelige genetiske abnormitet i mennesker embryonale aneuploidi 3. Aneuploidi er defineret som afvigelse (gevinst eller tab) af det rigtige antal kromosomer i en celle. Fænomenet med aneuploidi i menneskelige embryoner er fælles og øger med avanceret moderens alder 4,5. Fire randomiserede kontrollerede forsøg har fremhævet fordelen af at vælge kun chromosomally normal (euploid) embryoner til uterin overførsel, fordi denne strategi resulterede i øget implantation satser, lavere abort priser og en kortere tid for at opnå graviditet 6,7,8,9. Derfor kan forståelse ætiologien af menneskelige aneuploidi have stor betydning i assisteret reproduktion.
Selv om præ-implantation genetisk testning for aneuploidies er gavnligt i barnløshed behandlinger, mangler stadig en grundig forståelse af hvordan aneuploidies stammer. Det er almindeligt accepteret, at der er en positiv korrelation af meiotiske aneuploidies (opstod i gamet produktion) og moderens alder, men nogle kvinder nuværende embryonale aneuploidi priser, der afviger fra den gennemsnitlige sats for deres given alder 4. Disse tilfælde foreslå, at alder alene ikke er altid intelligent af risikoen for at generere et aneuploid Foster. Andre faktorer kan spille en rolle i at øge risikoen for embryonale aneuploidi, som gen-varianter.
Et centralt aspekt af efterforske det potentielle bidrag fra et gen variant til aneuploidi under oocyt meiose er at designe et system hurtigt vurdere meiotiske genfunktion. På grund af etiske begrænsninger og begrænset adgang er det upraktisk at udføre disse eksperimenter ved hjælp af menneskelige æg. Disse spørgsmål kan omgås ved hjælp af musen oocytter, og her en serie af assays til at vurdere menneskeligt genfunktion under meiose jeg er beskrevet. Af microinjecting messenger RNA (mRNA) kodning for genet variant af interesse, kan lokalisering af det menneskelige protein i mus æg visualiseres og bruges til at bestemme, hvis den Ektopisk udtryk for den wild-type og muteret humant protein resulterer i nogen fænotypiske forandringer, der kan føre til aneuploidi. Disse fænotyper omfatter en stigning i mikrotubuli, der tillægger den forkert at søster kinetochore og manglende evne til at støtte kromosom justering på metafase meiose i. vigtigere, denne protokol kan bruges til at undersøge både gevinst og tab af funktion genetiske varianter ved at etablere særlige eksperimentelle betingelser at udfordre nøglebegivenheder i oocyt meiose såsom spindel bygning og kromosom justering 10.
Protocol
Representative Results
Discussion
På grund af den hurtige og stigende identifikation af menneskers genetiske varianter forbundet med sygdom, er det vigtigt, at systemerne er etableret for at evaluere deres biologiske betydning. Forstå protein funktion i menneskelige meiose skaber særlige udfordringer, fordi menneskelige oocyter er dyrebare og sjældne og menneskelige sædceller er ikke indstillet til genmanipulation. Musen oocyter er et pattedyr modelsystem værdifulde for evaluering meiotiske menneskegener funktion 10,</s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af forskning tilskud fra American Society for Reproductive Medicine og Charles og Johanna Busch Memorial Fund på Rutgers, The State University i NJ til K.S. A.L.N. blev støttet af en bevilling fra N.I.H. (F31 HD0989597).
Materials
| 0.2 mL Seal-Rite PCR tube | USA Scientific | 1602-4300 | |
| 1 kb DNA Ladder | Thermo Scientific | SM0313 | |
| 100 bp DNA Ladder | Thermo Scientific | SM0243 | |
| 6X DNA loading Dye | Thermo Scientific | R0611 | |
| 9-well glass spot plate | Thomas Scientific | 7812G17 | |
| Agarose | Sigma Aldrich | A9539 | |
| Albumin from bovine serum | Sigma-Aldrich | A3294 | |
| Alexa-fluor-568 conjugated anti-mouse IgG | Thermo Scientific | A21050 | 1:200 dilution |
| Alexa-fluor-633 conjugated anti-human IgG | Thermo Scientific | A21091 | 1:200 dilution |
| Ampicillin | VWR | AA0356 | |
| Anti-vibration table | Technical Manufacturing Corp | any standard model | |
| Anti-Acetylated Tubulin antibody | Sigma Aldrich | T7451 | 1:100 diution |
| Anti-centromeric (CREST) antibody | Antibodies Incorportated | 15-234 | 1:30 dilution |
| Barrier (Filter) Pipette tips | Thermo Scientific | AM12635 | Make sure compatable with your brand of pipettors. These are compatible with Eppendorf brand pipettors. |
| BD Difco Dehydrated Culture Media: LB Agar, Miller (Luria Bertani) | Fisher Scientific | DF0445-07-6 | |
| BD Difco Dehydrated Culture Media: LB Broth, Miller (Luria Bertani) | Fisher Scientific | DF0446-07-5 | |
| Capillary tubing | Sutter | B100-75-10 | |
| Center Well organ culture dish | VWR | 25381-141 | |
| CO2 tank | For incubator | ||
| Confocal microscope | Zeiss | any standard model | |
| Centrifuge (With cooling ability) | Thomas Scientific | any standard model | |
| Cover Glass 11 x 22 mm | Thomas Scientific | 6663F10 | |
| Coverslips | Thomas Scientific | 6663-F10 | thickness will vary for particular microscopes |
| DAPI | Sigma Aldrich | D9542 | |
| DEPC H20 | Life Technologies | AM9922 | |
| Digital Dry Bath | Thermo Scientific | 888700001 | |
| Easy A high fidelity cloning enzyme | Agilent | 600400 | For DNA cloning |
| Enzymes for linearizing pIVT | New England Biolabs | NdeI or KasI can be used | |
| Ethidium Bromide | Thermo Scientific | 155585011 | |
| Fluorescent Microscope | Any Fluorescent microscope may be used | ||
| Formaldehyde (37%) | Thermo Fisher Scientifc | 9311 | |
| Formaldehyde RNA loading dye | Ambion | 8552 | |
| Frosted Microscope Slides (Uncharged) 25X75 mm | Fisher Scientific | 12-544-3 | |
| Full Length cDNA Clones | Can be obtained from any vendor that supplies open reading frame clones | ||
| Gel electrophoresis apparatus | Bio-Rad | any standard model | |
| Glass Pasteur Pipets | Fisher Scientific | 13-678-200 | |
| Globin Forward Primer for pIVT Construct | 5'- GAA GCT CAG AAT AAA CGC -3'. Can be purchased from any company that generates custom oligonucelotides | ||
| Globin Reverse Primer for pIVT Construct | 5'- ATT CGG GTG TTC TTG AGG CTG G -3' Can be purchased from any company that generates custom oligonucelotides | ||
| Holding pipettes | Eppendorf | 930001015 | Vacutip |
| Humidified Chamber | Tupperware can be used | ||
| Illustra Ready-To-Go RT-PCR beads | GE Life Sciences | 27925901 | |
| Incubator | any standard model with CO2 and water jacketed technology | ||
| Inverted Microscope | Nikon instruments | Any Standard model | |
| Image J (NIH) Software | NIH | Image Analysis software | |
| Lid of 96 well plate | Nalgene Nunc International | 263339 | |
| Low Adhesion 0.5 mL microcentrifgue tube | USA Scientific | 1405-2600 | |
| MacVector | MacVector | Sequence analysis software | |
| MG132 | Selleckchem | S2619 | |
| Microscope slides | Fisher Scientific | 12-544-3 | |
| Millenium RNA Markers-Formaldehyde | Ambion | AM7151 | |
| Milrinone | Sigma-Aldrich | M4659 | Resuspend in DMSO at 2.5mM |
| Mineral Oil | Sigma-Aldrich | M5310 | Only used embryo-tested, sterile-filtered |
| Monastrol | Sigma-Aldrich | M8515 | Resuspend in DMSO at 100 mM |
| Mouthpiece | Biodiseno | MP-001-Y | |
| N2 tank | for antivibration table | ||
| Nail Polish; Clear | Any clear nailpolish can be used | ||
| NanoDrop Microvolume UV-Vis Spectrophotometer | Thermo Scientific | any standard model | |
| NorthernMax 10X Denaturing Gel Buffer | Life Technologies | AM8676 | |
| NorthernMax 10X Running buffer | Life Technologies | AM8671 | |
| NuPAGE MOPS SDS Running buffer | Thermo Scnentific | NP0001 | |
| Organ Culture Dish 60x15mm | Life Technologies | 08-772-12 | |
| Paraformaldehyde | Polysciences, Inc. | 577773 | |
| PCR Thermal Cycler | Thermo Fisher Scientific | 4484075 | |
| Petri Dish 139 mm | Thermo Fisher Scientifc | 501V | |
| Petri dish 35 mm | Thermo Fisher Scientifc | 121V | |
| Petri Dish 60 mm | Falcon BD | 351007 | |
| Picoinjector | XenoWorks Digital Microinjector | any standard model | |
| Pipette puller | Flaming-Brown Micropipette puller | Model P-1000 | |
| pIVT plasmid | AddGene | 32374 | Empty vector suitable for oocyte expression. |
| Pregnant Mare Serum Gonadotropin | Lee BioSolutions | 493-10 | |
| QIAprep Spin Miniprep Kit | Qiagen | 27104 | purification of up to 20 uL of plasmid DNA |
| QIAquick PCR purification kit | Qiagen | 28104 | |
| Quikchange II site directed mutagenesis kit | Agilent | 200523 | mutagenesis kit for insertions and deletions |
| Quikchange lightning multi-site directed mutagenesis kit | Agilent | 210512 | mutagenesis kit for single site changes |
| Scissors (Fine point) | Fine science tools | 14393 | |
| Scissors (Medium point) | Fine science tools | WP114225 | |
| Seal-Rite 1.5 mL microcentrifuge tube | USA Scientific | 1615-5500 | |
| Slide Warmer | any standard model | ||
| Spectrophotometer (Nanodrop) | Thermo Fisher Scientific | ND-ONE-W | |
| Stereomicroscope | any standard model | ||
| Subcloning Efficiency DH5a Competent Cells | Thermo Fisher Scientifc | 18265017 | |
| Syringe | BD Bioscienes | 309623 | 1 ml, 27G(1/2) |
| T4 DNA Ligase | New England Biolabs | M0202L | |
| T7 mMessage Machine high-yield capped RNA transcription kit | Life Technologies | AM1340 | |
| TritonX-100 | Sigma-Aldrich | x-100 | |
| Tween-20 | Sigma-Aldrich | 274348 | |
| Tweezer (Fine point- Size 5) | Fine science tools | SN.743.12.1 | |
| UltraPure Dnase/Rnase-Free Distilled Water | Thermo Fisher Scientifc | 10977015 | |
| UltraPure Ethidium Bromide 10mg/mL | Thermo Fisher Scientifc | 15585011 | |
| UVP UV/White lite transilluminator | Fisher Scientific | UV95041501 | |
| Vectashield Mounting Medium | Vector Laboratories | H-1000 |
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