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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Published: November 1st, 2018

DOI:

10.3791/58327

1Neurobiology Laboratory, National Institute of Environmental Health Sciences, 2Comparative Medicine Branch, National Institute of Environmental Health Sciences, 3Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, 4Signal Transduction Laboratory, National Institute of Environmental Health Sciences
* These authors contributed equally

Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice.

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

This method provides detailed instructions for permeabilizing the zona pellucida of mouse fertilized eggs to make embryonic cells accessible for gene delivery by lentiviruses. Lentiviruses are designed by nature for efficient gene delivery to mammalian cells. They infect dividing and non-dividing cells and integrate the lentiviral genome into their host chromosomes1. The range of lentiviral host cells is readily expanded by pseudotyping the recombinant lentivirus with the vesicular stomatitis virus glycoprotein (VSV-G), due to the broad tropism of the VSV-G protein2. Following transduction, lentiviral genes are stably in....

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All animal procedures and treatments used in this protocol were in compliance with the NIH/NIEHS animal care guidelines and were approved by the Animal Care and Use Committee (ACUC) at the NIH/NIEHS, Animal Protocol 2010-0004.

1. Preparations

  1. Prepare/purchase recombinant non-propagating lentiviruses carrying your gene of interest. In this study, lentivirus SBI511 expressing copepod green fluorescent protein (copGFP; abbreviated to GFP) from an elongation factor 1a promoter was used.......

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Development of isolated/transduced mouse fertilized eggs can be checked under the microscope daily (Figure 1). Healthy embryos develop into blastocyst within 3–4 days. In this protocol, 60–70% of untreated embryos develop into blastocyst23. Out of 114 laser-perforated transduced embryos, 54 developed into blastocyst (rate of 47%) and 46 blastocysts expressed GFP (46/54=85%)23.

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The ability of the lentiviruses to integrate into their host genome makes them an ideal vector for stable gene delivery. Lentiviral vectors can carry up to 8.5 kilobase pair (kbp) of genetic material that can accommodate cell-specific or inducible promoters, selection markers, or fluorescent moieties. Incorporated genomic material can replicate as part of their host genome and be regulated to express or deactivate at desired time points. These vectors allow for spatiotemporal control over gene expression at various stage.......

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This research was supported by the Intramural Research Program of the National Institute of Health (NIH), National Institute of Environmental Health Sciences (NIEHS). We are grateful to Dr. Robert Petrovich and Dr. Jason Williams for critical reading of the manuscript and helpful advice. We would also like to acknowledge and thank Dr. Bernd Gloss, the Knockout core, the Flow Cytometry Facility, the Fluorescence Microscopy and Imaging Center, and the Comparative Medicine Branch facilities of the NIEHS for their technical contributions. We would like to thank Mr. David Goulding from the Comparative Medicine Branch and Ms. Lois Wyrick of the Imaging Center at the NIEHS f....

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Name Company Catalog Number Comments
CD510B-1 plasmid System Biosciences CD510B-1 used to package the lentivirus expressing EF1a-copGFP
Dimethylpolysiloxane Sigma DMPS5X culturing embryos
hyaluronidase Sigma H3506 used to remove cumulus cells
XYClone Laser Hamilton Thorne Biosciences perforating mouse fertilized eggs
Non-Surgical Embryo Transfer (NSET) Device ParaTechs 60010 NSET of embryos
KSOM medium Millipore MR-020P-5F culturing embryos
Composition of KSOM: mg/100mL
NaCl 555
KCl 18.5
KH2PO4 4.75
MgSO4 7H2O 4.95
CaCl2 2H2O 25
NaHCO3 210
Glucose 3.6
Na-Pyruvate 2.2
DL-Lactic Acid, sodium salt 0.174mL
10mM EDTA 100µL
Streptomycin 5
Penicillin 6.3
0.5% phenol red 0.1mL
L-Glutamine 14.6
MEM Essential Amino Acids 1mL
MEM Non-essential AA 0.5mL
BSA 100
M2 medium Millipore MR-015-D culturing embryos
Composition of M2: mg/100mL
Calcium Chloride 25.1
Magnesium Sulfate (anhydrous) 16.5
Potassium Chloride 35.6
Potassium Phosphate, Monobasic 16.2
Sodium Bicarbonate 35
Sodium Chloride 553.2
Albumin, Bovine Fraction 400
D-Glucose 100
Na-HEPES 54.3
Phenol Red 1.1
Pyruvic Acid 3.6
DL-Lactic Acid 295

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