Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

Summary

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

Abstract

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Introduction

The pioneering work of Gordon et al. in 1980 showed that after implantation in pseudopregnant mice, the plasmid DNA injection into the male pronuclei of fertilized oocytes can yield the production of transgenic animals that integrated the plasmid DNA¹. The demonstration that transgenic mammals can be generated had an enormous impact on global life sciences, opening the way to novel fields of research both for basic sciences and translational biomedical sciences. In the past four decades, DNA microinjection has become a routine practice. Although an enormous number of transgenic mice have been produced, the standard method is n....

Protocol

All procedures that include animal work have obtained ethical approval and have been authorized by the French Ministry of Research and Education under number APAFIS#5094-20 16032916219274 v6 and 05311.02. The ICM animal facility PHENOPARC has been accredited by the French Ministry of Agriculture under the accreditation number B75 13 19. The overall protocol requires performing each procedure within a precise time frame that is summarized in in Figure 1.

1. Animal Purchase and Preparation of Basic Compounds

1. Animal purchase
   1. Order 25 vasectomized males B6CBAF1/JRj that are 8 weeks of age (F....

Results

Transgenic animals were generated using the protocol presented here. Representative results both ubiquitous and cell type specific transgene expression are illustrated.

Constitutive expression of transgenes

Ubiquitous promoters are basic research tools to express transgenes in a sustained and efficient manner. Such promoters are used for a very large v.......

Discussion

The perivitelline injection of lentiviral vectors in fertilized oocytes described here resulted in the production of transgenic embryos that yielded more than 70% of transgenic embryos relative to total number of collected embryos. This result is consistent with previous reports and exemplifies the specificity of the procedure^2,7,10,11,12. When comparing all t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PMSG 50UI	Sigma	G4527
hCG 5000UI	Sigma	CG5-1VL
NaCl	Sigma	7982
100 mm petri dish	Dutsher	353003
4 wells Nunc dish	Dutsher	56469	IVF dish
M2 medium	Sigma	M7167
M16 medium	Sigma	M7292
0,22 µm Syringe filter	Dutsher	146611
Hyaluronidase Enzyme 30mg	Sigma	H4272	mouse embryo tested
Insulin serynge	VWR	613-3867	Terumo Myjector
Curved forceps	Moria	2183
Curved scissors	Moria	MC26
Aspirator tube assemblies for calibrated microcapillary pipettes	Sigma	A5177-5EA
Borosilicate glass capillaries	Harvard apparatus	GC 100-10
Horizontal micropipette puller	Narishige	PN-30
Microforge	Narishige	MF-900
Inverted microscope	Nikon	Transferman NK2 5188	Hoffman modulation contrast illumination is required
Micromanipulator	Eppendorf	Celltram air
Controler of holding pipet	Eppendorf	Femtojet
Mineral oil	Sigma	M8410	mouse embryo tested
Microinjector	Eppendorf	Femtojet	Can be used to inject DNA or viral vectors
Dumont # 5 forceps	Moria	MC 40
vannas micro scissors	Moria	9600
Isoflurane	centravet	ISO005	ISO-VET 100% 250ml
ocrygel	centravet	OCR002
Povidone iodure	centravet	VET001	vetedine 120ml
Buprenorphine	centravet	BUP002	Buprecare 0,3Mg/ml 10ml
Tris-HCl	Sigma	T5941	Trizma hydrochloride
EDTA	Sigma	E9884
SDS	Sigma	436143
NaCl	Sigma	S7653	powder
proteinase K	Sigma	P2308
oneTaq kit	NEB	M0480L
Primers	Eurogentec
Strip of 8 PCR tube	4titude	4ti-0781
96 well thermal cycler	Applied Biosystems	4375786	Veriti
Genomic DNA mini kit	invitrogen	K1820-02
Nanodrop 2000	Thermo Scientific	ND-2000C
qPCR Master mix	Roche	4887352001	SYBR Green
Multiwell plate 384	Roche	5217555001
qPCR instrument 384 well	Roche	5015243001	LightCycler 480