Generation of Mammalian Offspring by Haploid Embryonic Stem Cells Microinjection

Ling Shuai1, Wei Li1, Haifeng Wan1, Xiao‐Yang Zhao1, Liu Wang1, Qi Zhou1

1 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1A.6
DOI:  10.1002/9780470151808.sc01a06s31
Online Posting Date:  November, 2014
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Abstract

In this unit we introduce the derivation and genetic modification of mouse haploid embryonic stem (ES) cells. We detail how to produce haploid embryos and the subsequent ES derivation and cell culture. We further introduce readers to the intracytoplasmic injection processes of two types of haploid ES cells [androgenetic haploid ES (ahES) and parthenogenetic ES (phES)], both of which possess potential to produce fertile progenies by microinjection. This unit will be interesting to researchers who focus on recessive screens and transgenic animal model production with haploid stem cells. © 2014 by John Wiley & Sons, Inc.

Keywords: haploid embryonic stem cells; microinjection; genetic modification; recessive screens

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Production of Haploid Embryos and Derivation of Haploid Embryonic Stem Cells
  • Support Protocol 1: Purification of Haploid ES Cells by Flow Cytometric Sorting
  • Support Protocol 2: Gene Manipulation of Haploid ES Cells
  • Support Protocol 3: Production of Fertile Mice by Microinjection of Haploid ES Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Production of Haploid Embryos and Derivation of Haploid Embryonic Stem Cells

  Additional Materials (also see the protocol 1Basic Protocol)
  • Hoechst33342 (Invitrogen, cat. no. H3570)
  • Verapamil (Sigma, cat. no. V4629)
  • 1× phosphate‐buffered saline (PBS) Ca2+ and Mg2+ free (Invitrogen, cat. no. C14190)
  • Ice
  • 37°C water bath
  • 40‐μm cell strainer (BD Biosciences, cat. no. 352340)
  • 5‐ml FACS tubes (BD Biosciences)
  • Flow cytometer (BD Biosciences, FACSAriaII)

Support Protocol 1: Purification of Haploid ES Cells by Flow Cytometric Sorting

  Additional Materials (also see protocol 1Basic Protocol)
  • FACS‐enriched haploid cells (see protocol 2)
  • Hypoosmolar buffer (Eppendorf, cat.no. 940002001)
  • Plasmid carrying a PGK‐neo cassette (Addgene, Plasmid no. 13443, PGKneolox2DTA)
  • Mitomycin C‐treated MEF cells (see Bryja et al., ) from G418‐resistant mice [JAX Mice Strain 002356, C57BL/6J‐Tg(pPGKneobpA)3Ems/J]
  • G418, Geneticin (Invitrogen, cat. no. 11811‐031)
  • 4‐mm width cuvette
  • Transfection system (Eppendorf, Multiporator)
  • Mouth‐controlled glass pipet

Support Protocol 2: Gene Manipulation of Haploid ES Cells

  Additional Materials (also see the protocol 1Basic Protocol)
  • Mice: CD‐1 background female mice and 129Sv/Jae background male mice.
  • Stereoscope (ZEISS, SteREO Discovery.V20)
  • Mouth‐controlled glass pipet
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Figures

Videos

Literature Cited

Literature Cited
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