Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos
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/ul] 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/ul] 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.
id |
KOHA-OAI-AGRO:46849 |
---|---|
record_format |
koha |
institution |
UBA FA |
collection |
Koha |
country |
Argentina |
countrycode |
AR |
component |
Bibliográfico |
access |
En linea En linea |
databasecode |
cat-ceiba |
tag |
biblioteca |
region |
America del Sur |
libraryname |
Biblioteca Central FAUBA |
language |
eng |
topic |
CLONED CATTLE PARTHENOGENESIS GFP TRANSGENESIS ENHANCED GREEN FLUORESCENT PROTEIN GREEN FLUORESCENT PROTEIN IONOMYCIN ANIMAL ANIMAL EMBRYO CATTLE CELL NUCLEUS TRANSPLANTATION CONFOCAL MICROSCOPY CULTURE MEDIUM CUMULUS CELL CYTOLOGY CYTOPLASM DRUG EFFECT EMBRYO CULTURE FLUORESCENCE IN SITU HYBRIDIZATION GENE EXPRESSION GENE EXPRESSION PROFILING GENE TRANSFER GENETICS METABOLISM METHODOLOGY MICROINJECTION OOCYTE PARTHENOGENESIS PLASMID TIME ANIMALS CULTURE MEDIA CUMULUS CELLS CYTOPLASM DNA EMBRYO CULTURE TECHNIQUES EMBRYO, MAMMALIAN FERTILIZATION IN VITRO GENE EXPRESSION GENE EXPRESSION PROFILING GENE TRANSFER TECHNIQUES GREEN FLUORESCENT PROTEINS IN SITU HYBRIDIZATION, FLUORESCENCE IONOMYCIN MICROINJECTIONS MICROSCOPY, CONFOCAL NUCLEAR TRANSFER TECHNIQUES OOCYTES PARTHENOGENESIS PLASMIDS TIME FACTORS BOS BOVINAE MAMMALIA CLONED CATTLE PARTHENOGENESIS GFP TRANSGENESIS ENHANCED GREEN FLUORESCENT PROTEIN GREEN FLUORESCENT PROTEIN IONOMYCIN ANIMAL ANIMAL EMBRYO CATTLE CELL NUCLEUS TRANSPLANTATION CONFOCAL MICROSCOPY CULTURE MEDIUM CUMULUS CELL CYTOLOGY CYTOPLASM DRUG EFFECT EMBRYO CULTURE FLUORESCENCE IN SITU HYBRIDIZATION GENE EXPRESSION GENE EXPRESSION PROFILING GENE TRANSFER GENETICS METABOLISM METHODOLOGY MICROINJECTION OOCYTE PARTHENOGENESIS PLASMID TIME ANIMALS CULTURE MEDIA CUMULUS CELLS CYTOPLASM DNA EMBRYO CULTURE TECHNIQUES EMBRYO, MAMMALIAN FERTILIZATION IN VITRO GENE EXPRESSION GENE EXPRESSION PROFILING GENE TRANSFER TECHNIQUES GREEN FLUORESCENT PROTEINS IN SITU HYBRIDIZATION, FLUORESCENCE IONOMYCIN MICROINJECTIONS MICROSCOPY, CONFOCAL NUCLEAR TRANSFER TECHNIQUES OOCYTES PARTHENOGENESIS PLASMIDS TIME FACTORS BOS BOVINAE MAMMALIA |
spellingShingle |
CLONED CATTLE PARTHENOGENESIS GFP TRANSGENESIS ENHANCED GREEN FLUORESCENT PROTEIN GREEN FLUORESCENT PROTEIN IONOMYCIN ANIMAL ANIMAL EMBRYO CATTLE CELL NUCLEUS TRANSPLANTATION CONFOCAL MICROSCOPY CULTURE MEDIUM CUMULUS CELL CYTOLOGY CYTOPLASM DRUG EFFECT EMBRYO CULTURE FLUORESCENCE IN SITU HYBRIDIZATION GENE EXPRESSION GENE EXPRESSION PROFILING GENE TRANSFER GENETICS METABOLISM METHODOLOGY MICROINJECTION OOCYTE PARTHENOGENESIS PLASMID TIME ANIMALS CULTURE MEDIA CUMULUS CELLS CYTOPLASM DNA EMBRYO CULTURE TECHNIQUES EMBRYO, MAMMALIAN FERTILIZATION IN VITRO GENE EXPRESSION GENE EXPRESSION PROFILING GENE TRANSFER TECHNIQUES GREEN FLUORESCENT PROTEINS IN SITU HYBRIDIZATION, FLUORESCENCE IONOMYCIN MICROINJECTIONS MICROSCOPY, CONFOCAL NUCLEAR TRANSFER TECHNIQUES OOCYTES PARTHENOGENESIS PLASMIDS TIME FACTORS BOS BOVINAE MAMMALIA CLONED CATTLE PARTHENOGENESIS GFP TRANSGENESIS ENHANCED GREEN FLUORESCENT PROTEIN GREEN FLUORESCENT PROTEIN IONOMYCIN ANIMAL ANIMAL EMBRYO CATTLE CELL NUCLEUS TRANSPLANTATION CONFOCAL MICROSCOPY CULTURE MEDIUM CUMULUS CELL CYTOLOGY CYTOPLASM DRUG EFFECT EMBRYO CULTURE FLUORESCENCE IN SITU HYBRIDIZATION GENE EXPRESSION GENE EXPRESSION PROFILING GENE TRANSFER GENETICS METABOLISM METHODOLOGY MICROINJECTION OOCYTE PARTHENOGENESIS PLASMID TIME ANIMALS CULTURE MEDIA CUMULUS CELLS CYTOPLASM DNA EMBRYO CULTURE TECHNIQUES EMBRYO, MAMMALIAN FERTILIZATION IN VITRO GENE EXPRESSION GENE EXPRESSION PROFILING GENE TRANSFER TECHNIQUES GREEN FLUORESCENT PROTEINS IN SITU HYBRIDIZATION, FLUORESCENCE IONOMYCIN MICROINJECTIONS MICROSCOPY, CONFOCAL NUCLEAR TRANSFER TECHNIQUES OOCYTES PARTHENOGENESIS PLASMIDS TIME FACTORS BOS BOVINAE MAMMALIA Pereyra Bonnet, Federico Bevacqua, Romina Jimena La Rosa, Isabel Sipowicz, Pablo Radrizzani Helguera, Martín Fernández Martín, Rafael Salamone, Daniel Felipe Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos |
description |
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/ul] 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/ul] 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. |
format |
Texto |
topic_facet |
CLONED CATTLE PARTHENOGENESIS GFP TRANSGENESIS ENHANCED GREEN FLUORESCENT PROTEIN GREEN FLUORESCENT PROTEIN IONOMYCIN ANIMAL ANIMAL EMBRYO CATTLE CELL NUCLEUS TRANSPLANTATION CONFOCAL MICROSCOPY CULTURE MEDIUM CUMULUS CELL CYTOLOGY CYTOPLASM DRUG EFFECT EMBRYO CULTURE FLUORESCENCE IN SITU HYBRIDIZATION GENE EXPRESSION GENE EXPRESSION PROFILING GENE TRANSFER GENETICS METABOLISM METHODOLOGY MICROINJECTION OOCYTE PARTHENOGENESIS PLASMID TIME ANIMALS CULTURE MEDIA CUMULUS CELLS CYTOPLASM DNA EMBRYO CULTURE TECHNIQUES EMBRYO, MAMMALIAN FERTILIZATION IN VITRO GENE EXPRESSION GENE EXPRESSION PROFILING GENE TRANSFER TECHNIQUES GREEN FLUORESCENT PROTEINS IN SITU HYBRIDIZATION, FLUORESCENCE IONOMYCIN MICROINJECTIONS MICROSCOPY, CONFOCAL NUCLEAR TRANSFER TECHNIQUES OOCYTES PARTHENOGENESIS PLASMIDS TIME FACTORS BOS BOVINAE MAMMALIA |
author |
Pereyra Bonnet, Federico Bevacqua, Romina Jimena La Rosa, Isabel Sipowicz, Pablo Radrizzani Helguera, Martín Fernández Martín, Rafael Salamone, Daniel Felipe |
author_facet |
Pereyra Bonnet, Federico Bevacqua, Romina Jimena La Rosa, Isabel Sipowicz, Pablo Radrizzani Helguera, Martín Fernández Martín, Rafael Salamone, Daniel Felipe |
author_sort |
Pereyra Bonnet, Federico |
title |
Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos |
title_short |
Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos |
title_full |
Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos |
title_fullStr |
Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos |
title_full_unstemmed |
Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos |
title_sort |
novel methods to induce exogenous gene expression in scnt, parthenogenic and ivf preimplantation bovine embryos |
url |
http://ceiba.agro.uba.ar/cgi-bin/koha/opac-detail.pl?biblionumber=46849 |
work_keys_str_mv |
AT pereyrabonnetfederico novelmethodstoinduceexogenousgeneexpressioninscntparthenogenicandivfpreimplantationbovineembryos AT bevacquarominajimena novelmethodstoinduceexogenousgeneexpressioninscntparthenogenicandivfpreimplantationbovineembryos AT larosaisabel novelmethodstoinduceexogenousgeneexpressioninscntparthenogenicandivfpreimplantationbovineembryos AT sipowiczpablo novelmethodstoinduceexogenousgeneexpressioninscntparthenogenicandivfpreimplantationbovineembryos AT radrizzanihelgueramartin novelmethodstoinduceexogenousgeneexpressioninscntparthenogenicandivfpreimplantationbovineembryos AT fernandezmartinrafael novelmethodstoinduceexogenousgeneexpressioninscntparthenogenicandivfpreimplantationbovineembryos AT salamonedanielfelipe novelmethodstoinduceexogenousgeneexpressioninscntparthenogenicandivfpreimplantationbovineembryos |
_version_ |
1756046698324426752 |
spelling |
KOHA-OAI-AGRO:468492022-08-08T10:16:59Zhttp://ceiba.agro.uba.ar/cgi-bin/koha/opac-detail.pl?biblionumber=46849AAGNovel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryosPereyra Bonnet, FedericoBevacqua, Romina JimenaLa Rosa, IsabelSipowicz, PabloRadrizzani Helguera, Martín Fernández Martín, RafaelSalamone, Daniel Felipetextengapplication/pdfThe 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/ul] 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/ul] 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.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/ul] 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/ul] 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.CLONED CATTLE PARTHENOGENESISGFP TRANSGENESISENHANCED GREEN FLUORESCENT PROTEINGREEN FLUORESCENT PROTEINIONOMYCINANIMALANIMAL EMBRYOCATTLECELL NUCLEUS TRANSPLANTATIONCONFOCAL MICROSCOPYCULTURE MEDIUMCUMULUS CELLCYTOLOGYCYTOPLASMDRUG EFFECTEMBRYO CULTUREFLUORESCENCE IN SITU HYBRIDIZATIONGENE EXPRESSIONGENE EXPRESSION PROFILINGGENE TRANSFERGENETICSMETABOLISMMETHODOLOGYMICROINJECTIONOOCYTEPARTHENOGENESISPLASMIDTIMEANIMALSCULTURE MEDIACUMULUS CELLSCYTOPLASMDNAEMBRYO CULTURE TECHNIQUESEMBRYO, MAMMALIANFERTILIZATION IN VITROGENE EXPRESSIONGENE EXPRESSION PROFILINGGENE TRANSFER TECHNIQUESGREEN FLUORESCENT PROTEINSIN SITU HYBRIDIZATION, FLUORESCENCEIONOMYCINMICROINJECTIONSMICROSCOPY, CONFOCALNUCLEAR TRANSFER TECHNIQUESOOCYTESPARTHENOGENESISPLASMIDSTIME FACTORSBOSBOVINAEMAMMALIATransgenic Research |