Genetically Engineered Mice by Pronuclear DNA Microinjection

Janet L. DeMayo1, Jie Wang1, Dongcai Liang1, Ruina Zhang1, Francesco J. DeMayo1

1 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo110168
Online Posting Date:  September, 2012
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Abstract

The generation of transgenic mice by DNA microinjection is a powerful tool to investigate the molecular regulation of gene expression, development, and disease. The power of this technology is that foreign DNA can be introduced into every cell of a developing organism, and the phenotypic impact of this genetic modification can be investigated in a system under the constraints of normal development and physiology. The generation of transgenic mice requires the preparation of the transgene DNA construction, collection of one‐cell fertilized mouse embryos, injection of the transgene into mouse embryos, and transfer of the surviving embryos into a pseudopregnant female. Mice born from such manipulations are then screened for the presence of the transgene. The execution of these procedures requires a highly efficient system; otherwise, the cost of the generation of these mice can be cost prohibitive. However, the production of these animals can provide an invaluable research resource. Curr. Protoc. Mouse Biol. 2:245‐262 © 2012 by John Wiley & Sons, Inc.

Keywords: transgenic; mouse; DNA microinjection

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Transgene DNA
  • Basic Protocol 2: Embryo Collection
  • Basic Protocol 3: Microinjection
  • Basic Protocol 4: Embryo Transfer
  • Support Protocol 1: Embryo Culture
  • Basic Protocol 5: Identification of Transgenic Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Transgene DNA

  Materials
  • Isolated plasmid DNA or bacterial colony with BAC containing transgene of interest
  • Restriction enzyme(s) and appropriate reaction buffer(s)
  • TAE (see recipe)
  • Agarose (Thermo Fisher Scientific, #BP1356‐500)
  • 10 mg/ml ethidium bromide
  • GeneClean II kit (MP Biomedicals, www.mpbio.com)
  • Microinjection TE buffer (see recipe)
  • LB medium
  • Antibiotics (for growth of BAC culture)
  • P1 (50 mM Tris⋅Cl, 10 mM EDTA, pH 8.0). Store at 4°C
  • P2 (200 mM NaOH, 1% SDS). Store at room temperature
  • P3 (2.8 M potassium acetate, pH 5.5). Store at 4°C
  • Ready‐Lyse (Epicentre Biotechnologies)
  • NucleoBond BAC 100 kit (Clontech)
  • TE buffer, pH 7.5
  • 7.5 M potassium acetate, autoclaved
  • Proteinase K
  • 100% ethanol
  • RNase A
  • 10% SDS
  • Phenol, buffered, pH 8
  • Chloroform
  • 7.5 M ammonium acetate
  • Isopropanol
  • 70% ethanol
  • Terminase enzyme
  • 3.0 M sodium acetate
  • BAC microinjection buffer (see recipe)
  • Gel tray, gel box, and power supply for electrophoresis
  • Centrifuge bottles
  • JA‐10 rotor
  • High‐speed 30‐ml centrifuge tubes
  • Water bath, 30°C

Basic Protocol 2: Embryo Collection

  Materials
  • 3‐ to 4‐week‐old female mice
  • Pregnant mare serum gonadotropin (PMSG) (VWR International, #80056‐608)
  • hCG (purchase pregnyl hCG or substitute from local pharmacy)
  • Breeder male mice
  • M2 medium (Millipore, #MR‐015‐D)
  • Hyaluronidase (Sigma‐Aldrich, #H3506‐1G)
  • Microdissection tools
  • Embryo transfer pipets (see recipe)

Basic Protocol 3: Microinjection

  Materials
  • DNA from plasmid or BAC ( protocol 1) or lentiviral DNA
  • Paraffin oil
  • M2 medium (DNA injection) or M16 medium (Millipore #MR‐010P‐5F) (lentivirus injection)
  • Cytochalasin B stock solution (see recipe)
  • M2/CB (2.5 µl cytochalasin B stock solution in 497.5 µl M2 medium)
  • Mouse embryos ( protocol 2)
  • Dow Corning 200 Fluid, 50 CST (Ellsworth Adhesives, http://www.ellsworth.com)
  • Glass disposable micropipets, borosilicate with filament (Sutter Instrument Company #BF100‐78‐10)
  • Sutter micropipet puller, P1000 or P‐97
  • Teflon tubing
  • Injection and holding needle holders
  • Micrometer syringes for injection and holding needle holders (Stoelting)
  • Micromanipulators (Narishige or Eppendorf) – Coarse and hydraulic
  • Inverted microscope (e.g., Nikon, Zeiss)
  • Disposable micropipets (VWR)
  • Microforge (Sensaur)
  • 50 × 9‐mm petri dishes with tight lids (Fisher #08‐757‐105)

Basic Protocol 4: Embryo Transfer

  Materials
  • ICR females >24 g
  • B6D2F1 vasectomized breeder males
  • 1.25% Avertin working solution
  • Betadine
  • 70% ethanol
  • Syringe for administration of anesthetic
  • Hair clippers
  • Microdissection tools
  • Embryo transfer pipets (see recipe)
  • Mouth pipet and tubing for embryo transfers (Renova Life, Inc., #mp‐set and mp 25)
  • Dissecting microscope
  • Wound clips and applicator
  • Heating pad

Support Protocol 1: Embryo Culture

  Materials
  • Appropriate culture medium, such as KSOM, preincubated in CO 2 incubator
  • Silicon oil (Dow Corning 200 Fluid)
  • 60 × 15 mm culture dishes

Basic Protocol 5: Identification of Transgenic Mice

  Materials
  • 1.25% Avertin working solution
  • TNES (see recipe)
  • 20 mg/ml proteinase K
  • 5 M NaCl
  • 70% and 95% ethanol
  • TE, pH 7.5
  • Eartag or ear punch
  • 15‐ or 50‐ml test tubes
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Figures

Videos

Literature Cited

Literature Cited
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