Generation of Genetically Modified Mice through the Microinjection of Oocytes

Summary

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

Abstract

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

Introduction

Animal models, both in vertebrates and invertebrates, have been instrumental to examining the pathophysiology of human conditions such as Alzheimer's disease^1,2. They are also invaluable tools to search for disease modifiers and to ultimately develop novel treatment strategies in the hope of a cure. Although each model has intrinsic limitations, the use of animals as entire systemic models is vital to biomedical research. This is because the metabolic and complex physiological environment cannot be entirely simulated in tissue culture.

To date, the mouse remains the most common ....

Protocol

All procedures have been approved by the University of New South Wales Animals Care and Ethics Committee.

1. Preparation of the Transgene (Random Integration)

1. Analytical agarose gel electrophoresis.
   1. Digest the plasmid to excise the transgene using appropriate enzymes (1 h incubation) or fast-digest enzymes (15 to 30 min incubation) in a thermocycler following the manufacturer's recommendations (see Figure 2A and its legend).
   2. Cast a 1% tris-acetate-ethylenediaminetetraacetic acid (EDTA) (TEA) agarose gel, stained with 0.5-1.0 µg/mL ethidium bromide (Et....

Results

Below, the workflows for microinjection in the case of random integration and CRISPR-mediated gene targeting are described (Figure 1).

Figure 1: Typical Workflow.......

Discussion

Critical steps within the protocol

The generation of genetically modified mice is known to be technically challenging. However, the protocol presented here is an optimized and simplified method that allows one to master and troubleshoot the technique in record time. There are two steps necessary for the successful completion of the technique. First, the synthesis of linear DNA templates (for the synthesis of sgRNAs) can be achieved without magnesium chloride (MgCl₂). However, it is hig.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Micropipette 0.1-2.5 μL	Eppendorf	4920000016
Micropipette 2 - 20 μL	Eppendorf	4920000040
Micropipette 20 - 200 μL	Eppendorf	4920000067
Micropipette 100 - 1,000 μL	Eppendorf	4920000083
Molecular weight marker	Bioline	BIO-33025	HyperLadder 1 kb
Molecular weight marker	Bioline	BIO-33056	HyperLadder 100 bp
Agarose	Bioline	BIO-41025
EDTA buffer	Sigma-Aldrich	93296	10x - Dilute to 1x
Ethidium bromide	Thermo Fisher Scientific	15585011
SYBR Safe gel stain	Invitrogen	S33102
Gel extraction kit	Qiagen	28706
PCR purification kit (Qiaquick)	Qiagen	28106
Vacuum system (Manifold)	Promega	A7231
Nuclease-free microinjection buffer	Millipore	MR-095-10F
Ultrafree-MC microcentrifuge filter	Millipore	UFC30GV00
Cas9 mRNA	Sigma-Aldrich	CAS9MRNA
CRISPR expressing plasmid (px330)	Addgene	42230
Nuclease free water	Sigma-Aldrich	W4502
Phusion polymerase	New England Biolabs	M0530L
T7 Quick High Yield RNA kit	New England Biolabs	E2050S
RNA purification spin columns (NucAway)	Thermo Fisher Scientific	AM10070
ssOligos	Sigma-Aldrich	OLIGO STANDARD
Donor plasmid	Thermo Fisher Scientific	GeneArt
Hyaluronidase	Sigma-Aldrich	H3884
KSOMaa embryo culture medium	Zenith Biotech	ZEKS-100
Mineral oil	Zenith Biotech	ZSCO-100
M2 Medium	Sigma-Aldrich	M7167
Cytochalasin B	Sigma-Aldrich	C6762
Mouthpiece	Sigma-Aldrich	A5177
Glass microcapillaries	Sutter Instrument	BF100-78-10
Proteinase K	Applichem	A3830.0100
Dumont #5 forceps	Fine Science Tools	91150-20
Iris scissors	Fine Science Tools	91460-11
Vessel clamp	Fine Science Tools	18374-43
Wound clips	Fine Science Tools	12040-01
Clips applier	Fine Science Tools	12018-12
Micro-scissors	Fine Science Tools	15000-03
Cauterizer	Fine Science Tools	18000-00
Non-absorbable surgical sutures (Ethilon 3-0)	Ethicon	1691H
5% CO2 incubator	MG Scientific	Galaxy 14S
Spectrophotometer	Thermo Fisher Scientific	Nanodrop 2000c
Thermocycler	Eppendorf	6321 000.515
Electrophoresis set up	BioRad	1640300
UV Transilluminator	BioRad	1708110EDU
Thermocycler	Eppendorf	6334000069
Stereoscopic microscope	Olympus	SZX7
Inverted microscope	Olympus	IX71
2x Micromanipulators	Eppendorf	5188000.012
Oocytes manipulator	Eppendorf	5176000.025
Microinjector (Femtojet)	Eppendorf	5247000.013
Mice C57BL/6J strain	Australian BioResources	C57BL/6JAusb