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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This article aims to provide the methodology for lentiviral transgenesis in rat embryos using multiple injections of a virus suspension into the zygote perivitelline space. Female rats that are mated with a fertile male strain with a different dominant fur color is used to generate pseudopregnant foster mothers.

Abstract

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

Introduction

For many years, laboratory rodents, such as rats and mice, have been used to model human physiological and pathological conditions. Animal research has led to discoveries that were unattainable by any other means. Initially, genetic studies focused on the analysis of spontaneously occurring disorders and phenotypes that are considered to closely mimic the human condition1. The development of genetic engineering methods allowed the introduction or deletion of specific genes to obtain a desired phenotype. Therefore, the generation of transgenic animals is recognized as a fundamental technique in modern research that allows studies of gene functio....

Protocol

The production and application of viral vectors was in accordance with Biosafety Level 2 guidelines and was approved by the Polish Ministry of Environment. All experimental animal procedures that are described below were approved by the Local Ethical Committee. The animals were housed in individually ventilated cages at a stable temperature (21–23 °C) and humidity (50–60%) with ad libitum access to water and food under a 12 h/12 h light/dark cycle.

1. Lentiviral vector productio.......

Representative Results

Using the protocol described herein, lentiviral vectors that carried the Syn-TDP-43-eGFP construct were produced (physical LV titer = 3.4 x 108/µL) and then could be used for one-cell-stage embryo subzonal injections. Only embryos with two visible pronuclei were subjected to the procedure. The number of injections of viral suspensions was determined experimentally. High implantation efficiency and a simultaneous lack of transgenic offspring were considered indicators of an insufficient number of viral par.......

Discussion

Advances in transgenic technologies have made rodent models an invaluable tool in biomedical research. They provide the opportunity to study genotype-phenotype relationships in vivo. Here, we present a widely available alternative for conventional transgenesis by pronuclear injections. The use of lentiviral gene transduction bypasses the need for demanding microinjections because viral vectors can be injected under the zona pellucida. This approach does not affect embryo integrity, which essentially guarantees a 100% sur.......

Acknowledgements

This study was supported by the ANIMOD project within the Team Tech Core Facility Plus program of the Foundation for Polish Science, co-financed by the European Union under the European Regional Development Fund to WK.

....

Materials

NameCompanyCatalog NumberComments
7500 Real Time PCR SystemApplied Biosystems
Aerrane (isoflurane)BaxterFDG9623
Aspirator tube assemblies for calibrated microcapillary pipettesSigmaA5177-5EA
Atipam 5 mg/mlEurovet Animal Health BVN/A0.5 mg/kg
Baytril 25 mg/ml (enrofloksacin)BayerN/A5-10 mg/kg
Borosilicate glass capillaries with filament GC100TF-15Harvard Apparatus Limited30-0039injection capillary
Bupivacaine 25 mg/mlAdvanz PharmaN/A0.25% in  0.9% NaCl
Butomidor 10 mg/ml (butorphanol  tartrate)Orion PharmaN/A1 mg/kg
CELLSTAR Tissue Cell Culture Dish 35-mmGreiner Bio-One627160
CELLSTAR Tissue Cell Culture Dish 60-mmGreiner Bio-One628160
CellTram OilEppendorf5176 000.025
Cepetor (Medetomidine) 1 mg/mlcp-pharmaN/A0.5 mg/kg
Chorulon, Human Chorionic Gonadotrophin IntervetN/A150 IU/ ml ml 0.9% NaCl
DMEM low glucoseSigma AldrichD6048
DNase, RNase-freeA&A Biotechnology1009-100
EmbryoMax Filtered Light Mineral OilSigmaES-005-C
Envelope protein coding plasmid for lentiviral vectors (VSVg plasmid) ADDGENE14888
FemtoJetEppendorf4i /5252 000.013
Fetal Bovine SerumSigma AldrichF9665-500ML
Folligon, Pregnant Mare’s Serum Gonadotropin IntervetN/A125 IU/ml in .9% NaCl
HEK 293T cells ATCCATCC CRL-3216
Hyaluronidase from Bovine Testis SigmaH4272-30MG0.5 mg/ml in M2 medium
Inverted Microscope ZeissAxiovert 200
Ketamine 100mg/mlBiowet PulawyN/A50 mg/kg
Liquid ParaffinMerck Millipore8042-47-5
M16 medium EmbryoMaxSigmaMR-016-D
M2 mediumSigmaM7167
Magnesium Chloride 1MSigma Aldrich63069-100ML
MicroforgeNarishigeMF-900
Mineral OilSigmaM8410-500ML
NaCl 0.9%POLPHARMA OTCN/Asterile, 5ml ampules
Operation microscope Inami Ophthalmic InstrumentsDeca-21
Packaging system coding plasmid for lentiviral vectors  (delta R8.2 plasmid)ADDGENE12263
PEI reagent (Polyethylenimine, Mw ~ 25,000,),Polysciences, Inc23966-1
Penicilin-streptomycinSigma AldrichP0781-100ML
Phosphate Buffered Saline, pH 7.4, liquid, sterile-filtered, suitable for cell cultureSigma Aldrich806552-500ML
Puller Sutter Instrument Co.P-97
Reflex Clip Applier/Reflex ClipsWorld Precision Instruments500345/500346
Safil, polyglycolic acid, braided, coated, absorbable threadsB.Braun Surgical1048029
StereomicroscopeOlympusSZX16
Surgical Sewing ThreadB.Braun C1048040
SYBR Green PCR Master MixApplied Biosystem4334973
Tolfedine 4% (tolfenamic acid)VetoquinolN/A2 mg/kg
TransferMan NK2EppendorfN/A
Trypsin EDTA solutionSigma AldrichT3924-500ML
UltracentrifugeBeckman CoulterOptima L-100 XP 
VacuTipEppendorf5175108.000holders capillary
Vita-POSUrsapharmN/Aeye ointment
Warming PlateSemicN/A
Watchmaker ForcepsVWR470018-868

References

  1. Lazar, J., Moreno, C., Jacob, H. J., Kwitek, A. E. Impact of genomics on research in the rat. Genome Research. 15 (12), 1717-1728 (2005).
  2. Tarkowski, A. K. Studies on mouse chimeras developed from eggs fused in vitro.

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