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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.
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.
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....
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.......
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.......
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.......
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.
....Name | Company | Catalog Number | Comments |
7500 Real Time PCR System | Applied Biosystems | ||
Aerrane (isoflurane) | Baxter | FDG9623 | |
Aspirator tube assemblies for calibrated microcapillary pipettes | Sigma | A5177-5EA | |
Atipam 5 mg/ml | Eurovet Animal Health BV | N/A | 0.5 mg/kg |
Baytril 25 mg/ml (enrofloksacin) | Bayer | N/A | 5-10 mg/kg |
Borosilicate glass capillaries with filament GC100TF-15 | Harvard Apparatus Limited | 30-0039 | injection capillary |
Bupivacaine 25 mg/ml | Advanz Pharma | N/A | 0.25% in 0.9% NaCl |
Butomidor 10 mg/ml (butorphanol tartrate) | Orion Pharma | N/A | 1 mg/kg |
CELLSTAR Tissue Cell Culture Dish 35-mm | Greiner Bio-One | 627160 | |
CELLSTAR Tissue Cell Culture Dish 60-mm | Greiner Bio-One | 628160 | |
CellTram Oil | Eppendorf | 5176 000.025 | |
Cepetor (Medetomidine) 1 mg/ml | cp-pharma | N/A | 0.5 mg/kg |
Chorulon, Human Chorionic Gonadotrophin | Intervet | N/A | 150 IU/ ml ml 0.9% NaCl |
DMEM low glucose | Sigma Aldrich | D6048 | |
DNase, RNase-free | A&A Biotechnology | 1009-100 | |
EmbryoMax Filtered Light Mineral Oil | Sigma | ES-005-C | |
Envelope protein coding plasmid for lentiviral vectors (VSVg plasmid) | ADDGENE | 14888 | |
FemtoJet | Eppendorf | 4i /5252 000.013 | |
Fetal Bovine Serum | Sigma Aldrich | F9665-500ML | |
Folligon, Pregnant Mare’s Serum Gonadotropin | Intervet | N/A | 125 IU/ml in .9% NaCl |
HEK 293T cells | ATCC | ATCC CRL-3216 | |
Hyaluronidase from Bovine Testis | Sigma | H4272-30MG | 0.5 mg/ml in M2 medium |
Inverted Microscope | Zeiss | Axiovert 200 | |
Ketamine 100mg/ml | Biowet Pulawy | N/A | 50 mg/kg |
Liquid Paraffin | Merck Millipore | 8042-47-5 | |
M16 medium EmbryoMax | Sigma | MR-016-D | |
M2 medium | Sigma | M7167 | |
Magnesium Chloride 1M | Sigma Aldrich | 63069-100ML | |
Microforge | Narishige | MF-900 | |
Mineral Oil | Sigma | M8410-500ML | |
NaCl 0.9% | POLPHARMA OTC | N/A | sterile, 5ml ampules |
Operation microscope | Inami Ophthalmic Instruments | Deca-21 | |
Packaging system coding plasmid for lentiviral vectors (delta R8.2 plasmid) | ADDGENE | 12263 | |
PEI reagent (Polyethylenimine, Mw ~ 25,000,), | Polysciences, Inc | 23966-1 | |
Penicilin-streptomycin | Sigma Aldrich | P0781-100ML | |
Phosphate Buffered Saline, pH 7.4, liquid, sterile-filtered, suitable for cell culture | Sigma Aldrich | 806552-500ML | |
Puller | Sutter Instrument Co. | P-97 | |
Reflex Clip Applier/Reflex Clips | World Precision Instruments | 500345/500346 | |
Safil, polyglycolic acid, braided, coated, absorbable threads | B.Braun Surgical | 1048029 | |
Stereomicroscope | Olympus | SZX16 | |
Surgical Sewing Thread | B.Braun | C1048040 | |
SYBR Green PCR Master Mix | Applied Biosystem | 4334973 | |
Tolfedine 4% (tolfenamic acid) | Vetoquinol | N/A | 2 mg/kg |
TransferMan NK2 | Eppendorf | N/A | |
Trypsin EDTA solution | Sigma Aldrich | T3924-500ML | |
Ultracentrifuge | Beckman Coulter | Optima L-100 XP | |
VacuTip | Eppendorf | 5175108.000 | holders capillary |
Vita-POS | Ursapharm | N/A | eye ointment |
Warming Plate | Semic | N/A | |
Watchmaker Forceps | VWR | 470018-868 |
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