Establishment and Use of Mouse Haploid ES Cells

Martin Leeb1, Anthony C.F. Perry2, Anton Wutz3

1 Wellcome Trust‐Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, 2 Laboratory of Mammalian Molecular Embryology, Department of Biology and Biochemistry, University of Bath, Bath, 3 Institute of Molecular Health Sciences, Department of Biology, Swiss Federal Institute of Technology, ETH Zürich, Zürich
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo140214
Online Posting Date:  June, 2015
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Abstract

Haploid genetics has facilitated new insights into mammalian pathways and disease mechanisms. Most animal cells are diploid, and mammalian haploid cell cultures have remained elusive for a long time. Recent methodological progress has enabled the routine derivation of haploid stem cell lines from mammalian haploid embryos. Here we provide detailed protocols for the establishment, culture, and manipulation of parthenogenetic and androgenetic haploid embryonic stem cells from mouse embryos. © 2015 by John Wiley & Sons, Inc.

Keywords: embryonic stem cells; oocyte; haploid ES cells; parthenogenotes; androgenotes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Production of Haploid Mouse Embryos (Parthenogenotes) Through Activation of Oocytes
  • Alternate Protocol 1: Production of Haploid Embryos by Oocyte Activation using Ca2+‐Free Medium
  • Support Protocol 1: Preparation of Mouse Embryonic Fibroblast Feeder Cells
  • Basic Protocol 2: Haploid ES Cell Derivation from Haploid Mouse Embryos
  • Basic Protocol 3: Passaging Haploid ES Cell Cultures in 2i/LIF Medium Using Accutase
  • Alternate Protocol 2: Passaging ES Cell Cultures in 2i/LIF Medium with Trypsin
  • Basic Protocol 4: Purifying the Haploid G1 Population of ES Cells by Hoechst 33342 Sorting
  • Support Protocol 2: Analysis of DNA Content by Propidium Iodide Staining
  • Support Protocol 3: Cryostorage of Haploid ES Cell Lines
  • Support Protocol 4: Preparation of Haploid ES Cells for Blastocyst Injection
  • Support Protocol 5: Preparation and Mutagenesis of Haploid ES Cells for a Genetic Screening Experiment
  • Alternate Protocol 3: Production of Androgenetic Haploid Embryos
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Production of Haploid Mouse Embryos (Parthenogenotes) Through Activation of Oocytes

  Materials
  • Donor mice for oocyte collection
  • Pregnant mare serum gonadotropin (PMSG; see recipe)
  • Human chorionic gonadotropin (hCG; see recipe)
  • M2 medium (see recipe)
  • 100× hyaluronidase stock (see recipe)
  • KSOM medium (see recipe) or M16 medium (see recipe)
  • 100 mM SrCl 2 stock solution (dissolve in H 2O and filter sterilize through a 0.2‐μm filter; store in the dark at 4°C)
  • 0.5 M EGTA stock solution, pH 8: dissolve in H 2O and adjust to pH 8 using NaOH; filter sterilize through a 0.2‐μm filter
  • Mineral oil (Sigma, cat. no. M8410, embryo tested)
  • 10 mM CHIR99021 stock in DMSO (e.g., reagentsdirect cat no. 27‐H76, 20 mg; or Axon Medchem, cat. no. 1386)
  • PD0325901 (10 mM stock in DMSO, store at −20°C; e.g., reagentsdirect Cat No. 39‐C68, 20 mg; or Axon Medchem Cat. No. Axon 1408)
  • 2i/LIF medium (see recipe)
  • Stereomicroscope
  • Dumostar #5 fine biological forceps (Electron Microscopy Sciences, cat. no. 72705‐01)
  • 6‐cm IVF/organ culture dishes (e.g., Becton Dickinson, cat. no. 353038, or Corning, cat. no. 353653)
  • 6‐cm cell culture dishes
  • Transfer pipet assembly with glass barrel and silicone pipet housing (Drummond Scientific) either supplied separately as a Bulb Assembly Microcap (cat. no. 1‐000‐9000) or with 100‐μl glass Microcap capillaries (cat no. 1‐000‐1000)
  • Propane burner for pulling glass capillaries
  • Additional reagents and equipment for injection of mice (Donovan and Brown, )

Alternate Protocol 1: Production of Haploid Embryos by Oocyte Activation using Ca2+‐Free Medium

  Additional Materials (also see protocol 1)
  • Ca2+‐free CZB medium (see recipe)
  • 3.5‐cm culture dishes

Support Protocol 1: Preparation of Mouse Embryonic Fibroblast Feeder Cells

  Materials
  • Mouse embryos (embryonic day 13.5)
  • MEF‐DMEM medium (see recipe)
  • 0.25% trypsin/EDTA solution (see recipe)
  • Freezing medium: 80% (v/v) FBS (e.g., PAN Biotech, cat. no. P30‐1502)/20% (v/v) DMSO (filter through 0.2‐μm filter and cool on ice before use; always prepare fresh)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010‐015)
  • Scalpel
  • 5‐ml syringe with 16‐G needle
  • 15‐cm tissue culture dish
  • 50‐ml conical centrifuge tubes (e.g., Corning Falcon)
  • Gammacell 40 irradiator (Best Theratronics)
  • Centrifuge
  • Gelatin‐coated (see recipe) tissue culture flasks

Basic Protocol 2: Haploid ES Cell Derivation from Haploid Mouse Embryos

  Materials
  • Haploid mouse embryos ( protocol 1)
  • Acidic Tyrode's solution (Sigma, cat. no. T1788; store in 1‐ml aliquots at −20°C)
  • M2 medium (see recipe)
  • 2i medium (see recipe)
  • 0.25% trypsin/EDTA solution (see recipe)
  • ES‐DMEM medium with 15% FBS (see recipe)
  • ES cell culture medium with serum replacement (alternative to ES‐DMEM with 15% FBS; see recipe)
  • Mouth pipet for handling of embryos (Sigma‐Aldrich, cat. no. A5177; or a transfer pipet can be used, e.g., Drummond Scientific; also see protocol 1 materials list)
  • Glass capillaries (Blaubrand, 50 μl, BRAND, cat. no. 708733)
  • 96‐well cell culture plates (e.g., Nuclon, Thermo Scientific, cat. no. 167008), coated with gelatin (see recipe) and feeders ( protocol 3)
  • Vacuum source and Pasteur pipets
  • Eppendorf Repeater Pipette Plus
  • Combitips (Eppendorf: 1‐ml, cat. no. 0030089642; 2.5‐ml, cat. no. 0030089650; 5 ml, cat. no. 0030089669)
  • 200‐μl (P‐200) aerosol‐barrier pipet tips
  • 24‐well cell culture plates (e.g., Nuclon, Thermo Scientific, cat. no. 142475), coated with gelatin (see recipe) and feeders ( protocol 3)

Basic Protocol 3: Passaging Haploid ES Cell Cultures in 2i/LIF Medium Using Accutase

  Materials
  • Haploid ES cell culture ( protocol 4)
  • Accutase solution (e.g., Sigma, cat. no. A6964‐100 ML)
  • DMEM/F12 (Life Technologies, cat. no. 21331‐020) with 0.045% BSA [3 ml of 7.5% BSA solution (Life Technologies, cat. no. 15260‐037) per 500 ml medium]
  • 2i/LIF medium (see recipe)
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Centrifuge with swinging‐bucket rotor
  • Tissue culture plates and/or flasks, gelatin coated (see recipe)

Alternate Protocol 2: Passaging ES Cell Cultures in 2i/LIF Medium with Trypsin

  Additional Materials (also see protocol 5)
  • 0.25% trypsin/EDTA solution (see recipe)
  • ES‐DMEM medium with 15% FBS (see recipe)

Basic Protocol 4: Purifying the Haploid G1 Population of ES Cells by Hoechst 33342 Sorting

  Materials
  • 10 mg/ml Hoechst 33342 in H 2O (Life Technologies, cat. no. H3570)
  • ES‐DMEM medium with 15% FBS (see recipe)
  • Haploid ES cell cultures ( protocol 4)
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Falcon 70 μm cell strainer (Corning, cat. no. 352350)
  • Polypropylene FACS tube (Corning, cat. no. 352063)
  • Cell sorter: e.g., Beckman Coulter Moflo or Becton Dickinson FACS Aria equipped with an UV laser
  • Cell culture plates and flasks of appropriate size, gelatin (see recipe) and feeder ( protocol 3) coated
  • Additional reagents and equipment for dissociating haploid ES cell cultures ( protocol 5 or protocol 6)

Support Protocol 2: Analysis of DNA Content by Propidium Iodide Staining

  Materials
  • Haploid ES cell cultures ( protocol 4)
  • 70% ethanol, cold
  • Phosphate‐buffered saline (Life Technologies, cat. no. 10010‐015)
  • 100 μg/ml RNase A (e.g., Sigma, cat. no. R4642)
  • 50 mg/liter propidium iodide in PBS (store at 4°C protected from light)
  • Flow analyzer (e.g., Cyan ADP, Beckman Coulter)
  • Benchtop centrifuge

Support Protocol 3: Cryostorage of Haploid ES Cell Lines

  Materials
  • Haploid ES cell cultures ( protocol 4)
  • Freezing medium: 80% (v/v) FBS (e.g., PAN Biotech, cat. no. P30‐1502)/20% (v/v) DMSO (filter through 0.2‐μm filter and cool on ice before use; always make fresh)
  • Liquid N 2
  • ES‐DMEM medium with 15% FBS (see recipe)
  • Cryovials, e.g., Nunc CryoTube vial (Thermo Scientific, cat. no. 368632)
  • Freezing box
  • Liquid N 2 cell storage tank
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Cell culture plates and flasks of appropriate size, gelatin (see recipe) and feeder ( protocol 3) coated

Support Protocol 4: Preparation of Haploid ES Cells for Blastocyst Injection

  Materials
  • Haploid ES cell cultures ( protocol 4)
  • ES‐DMEM medium supplemented with 15% FBS (see recipe) with and without 20 mM HEPES (add from 1 M HEPES stock, pH 7.5; Life Technologies, cat. No.: 15630‐056)
  • 6‐well cell culture plates (e.g., Nuclon, Thermo Scientific, cat. no. 140675), coated with gelatin (see recipe)
  • Centrifuge
  • Additional reagents and equipment for preparing single‐cell suspension from haploid ES cell cultures ( protocol 5 or protocol 6)

Support Protocol 5: Preparation and Mutagenesis of Haploid ES Cells for a Genetic Screening Experiment

  Materials
  • Haploid ES cell cultures ( protocol 4)
  • 0.25% trypsin/EDTA solution (see recipe)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010‐015)
  • Gene‐trap piggyBac transposon (e.g., Sanger plasmid repository; http://www.sanger.ac.uk/resources/)
  • Hyperactive transposase (Yusa et al., ; (e.g., Sanger plasmid repository; http://www.sanger.ac.uk/resources/))
  • 75‐cm2 (T‐75) tissue culture flasks
  • GenePulser Xcell Electroporation system (BioRad) or equivalent
  • 4‐mm electroporation cuvettes
  • 15‐cm tissue culture plates
  • Additional reagents and equipment for trypsinization of cells (see protocol 5 or protocol 6)

Alternate Protocol 3: Production of Androgenetic Haploid Embryos

  Materials
  • PVP 360 solution: Polyvinylpyrrolidone (PVP 360, average M r≈360,000) at 5% to 20% (w/v; typically ∼10%) dissolved in sterile, high‐quality distilled water, and filter‐sterilized through a 0.45‐μm filter (store in 1‐ml aliquots at −20°C, with one thawed for current use that may be kept at room temperature for >1 month)
  • Cytochalasin B [ICN, cat. no. 195119; stock cytochalasin B may be stored in aliquots for several months at −20°C as a 1000× (5 mg/ml) solution in DMSO (Sigma, cat. no. D‐8418)
  • M2 medium (EMD Millipore; Quinn et al., )
  • Mineral oil (Shire; Nakalai Tesque)
  • Oocytes (see protocol 1) derived from strain B6D2F 1 mice, a female C57BL/6 × male DBA/2 F 1 hybrid (suppliers include Charles River Laboratories and Shimizu Laboratory Supplies Co.)
  • KSOM medium (see recipe)
  • Mouse sperm suspension (∼104 sperm/ml)
  • Optilux 15 × 90–mm petri dishes (BD Falcon, cat. no. 351001 or 353003): bottoms are suitable for oocyte/embryo collection and lids for micromanipulation (compatible with Hoffman modulation optics)
  • 10 × 35–mm dishes (BD Falcon, cat. no. 35 1008) and 15 × 60–mm dishes (BD Falcon, cat. no. 35 1007) for embryo culture
  • Workstation comprising inverted microscope, such as IX71 (Olympus) equipped with MO‐202U micromanipulators, IM‐5 and/or ‐6 injectors (Narishige), and Hoffman modulation contrast optics (4× and 20× objectives), or equivalent
  • Borosilicate glass capillaries (Sutter, cat. no. B100‐75‐100), 10 cm × 1 mm (outer diameter), 0.75 mm (inner diameter); these are used to pull injection and enucleation pipets (Yoshida and Perry, )
  • Microforge (manufacturers include Narishige and De Fonbrune).
  • Pipet puller such as the Flaming/Brown P97/IVF micropipet puller (Sutter Instrument Co.)
  • Holding pipets (Eppendorf)
  • Air‐cushioned table or platform (suppliers respectively include Technical Manufacturing Corporation and Meiritsu); the need for insulation against vibration depends on the location of the workstation; freezers, air‐conditioning, elevators and busy nearby roads are common causes of persistent vibration
  • Piezo‐actuated micromanipulator, such as the PMAS‐CT150 (Prime Tech)
  • Mouth pipet for handling of embryos [this can be made from a Microcap bulb dispenser (Drummond Scientific Company) equipped with an air‐tight assembly of silicone tubing attached to filtered blue Gilson tips and a pipet pulled by hand from 116‐mm glass capillaries (Drummond, cat no. 1‐000‐1000)]
  • Stereomicroscope, such as the SZX12 (Olympus)
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Literature Cited

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