Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine Transgene-Expressing Embryos.
The use of vesicles co-incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co-incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx-egfp was injected circular (CP) at 3, 30 and 300 ng/μl and linear (LP) at 30 ng/μl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/μl (N = 105), 30 ng/μl (N = 95) and 300 ng/μl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/μl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/μl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co-incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids.
Cátedra de Histología y Embriología, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina.
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Luchetti
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina.
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RJ
Bevacqua
Cátedra de Histología y Embriología, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina.
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Lorenzo
Cátedra de Histología y Embriología, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina.
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MF
Tello
Centro de Estudios Biomedicos, Biotecnologicos, Ambientales y Diagnostico (CEBBAD), Universidad Maimonides, Ciudad Autónoma de Buenos Aires, Argentina.
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Willis
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina.
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CP
Buemo
Cátedra de Histología y Embriología, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina.
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Lombardo
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina.
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Salamone
D000818:Animals; D030801:Animals, Genetically Modified; D053938:DNA Fragmentation; D046149:Embryo Culture Techniques / Q000662:veterinary*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D020287:In Situ Nick-End Labeling; D010063:Ovum / Q000502:physiology*; D010312:Parthenogenesis; D010957:Plasmids; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods / Q000662:veterinary*; D013552:Swine / Q000196:embryology*
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins