Expression of fluorescent reporter protein in equine embryos produced through intracytoplasmic sperm injection mediated gene transfer (ICSI-MGT).
Abstract: Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transgenic livestock with applications in biomedicine and agriculture. To date, two studies have reported the production of transgenic equine embryo with, however, low efficiency in blastocyst production and transgene expression. The aim of the present study was to develop a method which allowed the efficient production of transgene-expressing embryos of the equine species through SMGT. To overcome problems due to in vitro fertilization (IVF) in horses, the ICSI procedure was associated with SMGT. The uptake of exogenous DNA in equine spermatozoa was assessed using a spectrophotometric approach and its internalisation using real time PCR and confocal laser scanning microscopy (CLSM). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of eGFP and then for the transmission of the transgene. Our results suggested that the maximal uptake of exogenous DNA in equine spermatozoa occurs from 30 to 60min of co-incubation. Furthermore, real time PCR analysis suggested that the internalisation of exogenous DNA in the highest quality spermatozoa was slightly greater than in those having the poorest quality parameters. Confocal laser scanning microscopy analysis confirmed that exogenous DNA is internalised by membrane intact spermatozoa. In this study, 22 embryos were produced, 8 of which reached the 8-cell stage or greater. Our data confirmed the transmission of the transgene in 86.3% of the cleaved embryos and the expression of the transgene in 25% of the embryos. These data allowed us to affirm that this method is highly efficient in producing equine embryos which are able to express high levels of the exogenous protein.
Copyright © 2012 Elsevier B.V. All rights reserved.
Publication Date: 2012-12-26 PubMed ID: 23312469DOI: 10.1016/j.anireprosci.2012.12.010Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research explores the development of a method to produce genetically modified horse embryos using sperm-mediated gene transfer (SMGT), specifically through Intracytoplasmic sperm injection (ICSI), with the goal of efficaciously enabling the expression of foreign genes.
Methodology
- The study involved the observation of DNA uptake in horse sperm, using spectrophotometry and verify its internalization through Real-time Polymerase Chain Reaction (PCR) and Confocal Laser Scanning Microscopy (CLSM).
- Hindrances accompanying conventional in vitro fertilization (IVF) were circumvented by implementing ICSI in partnership with SMGT.
- The embryos resulting from this ICSI-SMGT procedure were then analyzed for the manifestation of the Enhanced Green Fluorescent Protein (eGFP), a commonly used reporter gene.
- Finally, the researchers checked the transfer of the altered gene from the sperm to the embryos.
Findings
- The study found that the optimal DNA uptake by horse sperm occurs between 30 to 60 minutes of co-incubation.
- The internalization of the foreign DNA was marginally higher in the healthiest sperm compared to sperm with lower quality parameters.
- Using CLSM, it was verified that the foreign DNA is indeed internalized by intact sperm membranes.
- Out of a total of 22 produced embryos, 8 developed up to or beyond the 8-cell stage.
- The researchers reported that the introduced gene was transmitted to a significant 86.3% of the cleaved embryos, with 25% of these embryos expressing the transgene.
Conclusion
- Overall, the study concluded that the ICSI-SMGT procedure demonstrated high efficiency in producing equine embryos that successfully express high levels of the introduced gene.
- This work has significant implications, paving the path for further development of techniques for producing transgenic horses, with broad potential applications in both the biomedical and agricultural fields.
Cite This Article
APA
Zaniboni A, Merlo B, Zannoni A, Bernardini C, Lavitrano M, Forni M, Mari G, Bacci ML.
(2012).
Expression of fluorescent reporter protein in equine embryos produced through intracytoplasmic sperm injection mediated gene transfer (ICSI-MGT).
Anim Reprod Sci, 137(1-2), 53-61.
https://doi.org/10.1016/j.anireprosci.2012.12.010 Publication
Researcher Affiliations
- Department of Veterinary Medical Sciences - DIMEVET, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano dell'Emilia, Bologna, Italy. Electronic address: andrea.zaniboni3@unibo.it.
MeSH Terms
- Animals
- Animals, Genetically Modified / physiology
- Embryo, Mammalian / physiology
- Embryo, Mammalian / ultrastructure
- Female
- Gene Transfer Techniques / veterinary
- Green Fluorescent Proteins / genetics
- Green Fluorescent Proteins / physiology
- Horses / genetics
- Horses / physiology
- Male
- Microscopy, Confocal / veterinary
- RNA / chemistry
- RNA / genetics
- Real-Time Polymerase Chain Reaction / veterinary
- Sperm Injections, Intracytoplasmic / methods
- Sperm Injections, Intracytoplasmic / veterinary
- Spermatozoa / physiology
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