title: Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos.
abstract: The import of exogenous DNA (eDNA) from the cytoplasm to the nucleus represents a key intracellular obstacle for efficient gene delivery in mammalian cells. In this study, cumulus cells or oolemma vesicles previously incubated with eDNA, and naked eDNA were injected into the cytoplasm of MII oocytes to evaluate their efficiency for eDNA expressing bovine embryo production. Our study evaluated the potential of short time co-incubation (5 min) of eDNA with; (1) cumulus cells, to be used as donor cells for SCNT and (2) oolemma vesicles (vesicles) to produce parthenogenic transgene expressing embryos. In addition, we included a group consisting of the injection of eDNA alone (plasmid) followed by parthenogenic activation. Two different pCX-EGFP plasmid concentrations (50 and 500 ng/μl) were employed. The results showed that embryos produced by SCNT and by vesicle injection assisted by chemical activation were able to express the eDNA in higher rates than embryos injected with plasmid alone. The lower plasmid concentration allowed the highest development rates in all groups. Using confocal microscopy, we analyzed the interaction of FITC- labeled eDNA with cumulus cells and vesicles as well as oocytes injected with labeled plasmid alone. Our images demonstrated that eDNA interacted with cumulus cells and vesicles, resulting an increase in its expression efficiency. In contrast, oocytes injected with DNA alone did not show signs of transgene accumulation, and their eDNA expression rates were lower. In a further experiment, we evaluated if transgene-expressing embryos could be produced by means of vesicle injection followed by IVF. The lower plasmid concentration (50 ng/μl) injected after IVF, produced the best results. Preliminary FISH analysis indicated detectable integration events in 1/5 of SCNT blastocysts treated. Our studies demonstrate for the first time that short term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT embryos and also that parthenogenic, eDNA- expressing embryos can be obtained by injection of vesicles or eDNA alone. Moreover, eDNA-expressing embryos can be also obtained by cytoplasmic injection vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection technique.
authors: Laboratorio de Biotecnologia Animal, Facultad de Agronomía, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors: Federico
authors: F
authors: Pereyra-Bonnet
authors: Romina
authors: R
authors: Bevacqua
authors: Isabel
authors: I
authors: La Rosa
authors: Pablo
authors: P
authors: Sipowicz
authors: Martin
authors: M
authors: Radrizzani
authors: Rafael
authors: R
authors: Fernandez-Martin
authors: Daniel
authors: D
authors: Salamone
mesh_terms: D000818:Animals; D002417:Cattle; D003470:Culture Media / Q000378:metabolism; D054885:Cumulus Cells / Q000166:cytology / Q000378:metabolism; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D046149:Embryo Culture Techniques / Q000379:methods*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005307:Fertilization in Vitro / Q000379:methods*; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence; D015759:Ionomycin / Q000494:pharmacology; D008845:Microinjections; D018613:Microscopy, Confocal; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010312:Parthenogenesis; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: D003470:Culture Media; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015759:Ionomycin; D004247:DNA