The fate of DNA injected into mammalian oocytes and zygotes at different stages of the cell cycle

Powell, D. J.; Galli, Cesare; Moor, R. M.

doi:10.1530/jrf.0.0950211

Cited by 16 publications

(9 citation statements)

References 18 publications

(23 reference statements)

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“…These results are in agreement with previous observations for IVF bovine embryos [4]. In experiment 3, covalently closed circular plasmids were used for complex formation since its linearized form could be more liable to degradation by nucleases present in the culture medium, or even in embryo cytoplasm before its delivery into the nucleus [28]. In this experiment, crotamine-DNA complexes were demonstrated to remain stably formed from a few minutes after their initial incubation until 24 h later in PBS medium.…”

Section: Discussionsupporting

confidence: 90%

Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression

Campelo

Canel

Bevacqua

et al. 2016

J Assist Reprod Genet

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Purpose Crotamine is capable of penetrating cells and embryos and transfecting cells with exogenous DNA. However, no studies are available regarding its uptake by parthenogenetic (PA) embryos or its use for transfection in in vitro fertilized (IVF) embryos. This study aimed to determine the translocation kinetics of crotamine into PA and IVF bovine embryos and assess its effect over in vitro development of PA embryos. Moreover, crotamine-DNA complexes were used to test the transfection ability of crotamine in bovine IVF zygotes. Methods PA and IVF embryos were exposed to labeled crotamine for four interval times. Embryo toxicity was assayed over PA embryos after 24 h of exposure to crotamine. Additionally, IVF embryos were exposed to or injected with a complex formed by crotamine and pCX-EGFP plasmid. Results Confocal images revealed that crotamine was uptaken by PA and IVF embryos as soon as 1 h after exposure. Crotamine exposure did not affect two to eight cells and blastocyst rates or blastocyst cell number (p > 0.05) of PA embryos. Regarding transfection, exposure or injection into the perivitelline space with crotamine-DNA complex did not result in transgeneexpressing embryos. Nevertheless, intracytoplasmic injection of plasmid alone showed higher expression rates than did injection with crotamine-DNA complex at days 4 and 7 (p < 0.05). Conclusions Crotamine is able to translocate through zona pellucida (ZP) of PA and IVF embryos within 1 h of exposure without impairing in vitro development. However, the use of crotamine does not improve exogenous DNA expression in cattle embryos, probably due to the tight complexation of DNA with crotamine.

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Section: Discussionsupporting

confidence: 90%

Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression

Campelo

Canel

Bevacqua

et al. 2016

J Assist Reprod Genet

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“…These ligation products are detected almost immediately after injection in mice (Burdon and Wall, 1993). In cattle the cytoplasm of pronuclear stage zygotes is very efficient in ligating DNA into large concatomers (Powell et al, 1992). During microinjection DNA leaks out of the pronucleus and into the cytoplasm (Chen et al, 1986).…”

Section: Discussionmentioning

confidence: 99%

Frequency of detection of exogenous DNA 7, 14, and 21 days after microinjection of bovine zygotes

Krisher

Gibbons

Gwazdauskas

et al. 1995

Animal Biotechnology

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The frequency of PCR positive detection of exogenous DNA was determined in bovine embryos over a 21 day period of culture after microinjection. After in vitro maturation and in vitro fertilization, embryos were microinjected with one of two genomic DNA constructs. Approximately 5% of the injected embryos were prepared for PCR analysis immediately after injection (d 0). After culture for 7 d on Buffalo Rat liver (BRL) cells, embryos were assessed for stage of development. Embryos developing to the morula or blastocyst stage were randomly assigned to two groups; PCR analysis (d 7) or extended culture. Extended culture embryos were cultured for an additional 7 d. Embryos maintaining a blastocoel at d 14 were considered alive, and were assigned randomly to either further culture or PCR analysis. Of zygotes examined after d 7 of culture, 26% (189/733) of control and 12% (294/2482) of injected embryos had developed to the morula or blastocyst stage (control vs. injected; P<0.01). After 14 d of culture, 33% (10/30) of control and 51% (100/196) of injected embryos were alive (P>0.05). After 21 d of culture, 29% (2/7) of control and 14% (9/66) of injected embryos were alive (P>0.05). Analysis of d 0 injected embryos resulted in a transgene detection frequency of 100% (46/46). At d 7, detection frequency was 82% (51/62) of morulae and blastocysts, and was 84% (513/610) of dead embryos. Transgene detection frequencies decreased to 49% (18/37) and 50% (41/82) on d 14, and 38% (3/8) and 22% (5/23) on d 21 in live and dead embryos, respectively. Transgene detection frequency between live embryos on d 0, 7, and 14 was significantly different (P<0.01). DNA detection frequency in microinjected bovine embryos by PCR analysis does not give a reliable indication of live transgenic birth rates, based on reported rates, before embryo transfer must be performed.

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“…Possibly DNA is integrated at the site of chromosomal breaks that occur as a result of pronuclear swelling during the microinjection process (Brinster et al, 1985). Powell et al (1992) found that pronuclear stage bovine embryos generate significant amounts of large ligation products when DNA is injected into the cytoplasm. This extrachromosomal recombination probably occurs as a result of opportunistic repair-ligation mechanisms (Bishop and Smith, 1989).…”

Section: Discussionmentioning

confidence: 99%

“…Cattle may not degrade DNA as readily as mice, because the proportion of positive mouse blastocysts is closer to the proportion of transgenic mice (Burdon and Wall, 1992;Canseco et al, 1994), compared with cattle. There are differences in the ability of bovine and porcine oocytes to ligate and degrade DNA (Powell et al, 1992). Bovine embryos may create such large ligation products that they are unable to degrade them as quickly as do mouse embryos.…”

Section: Discussionmentioning

confidence: 99%

Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos

Krisher

Gibbons

Canseco

et al. 1994

Transgenic Research

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The effect of DNA microinjection at various times after in vitro insemination on DNA detection and survival rates of bovine embryos was investigated. Oocytes were inseminated 24 h after maturation with frozen/thawed semen prepared with a Percoll separation procedure. At 11, 15 and 19 h after insemination, embryos were centrifuged to visualize pronuclei and microinjected with a murine whey acidic protein-human protein C genomic DNA construct. After culture for 7 days on Buffalo Rat Liver cells, embryos were assessed for stage of development and assayed for the presence of the transgene by polymerase chain reaction. Of zygotes in the 11 h after insemination treatment, 16% (25/152) of non-injected and 7% (11/161) of injected embryos developed to the morula or blastocyst stage. Comparable development of non-injected and injected embryos treated at 15 h after insemination was 15% (23/158) and 4% (6/159) and treated at 19 h after insemination was 14% (23/162) and 1% (1/165), respectively. Development of injected embryos was greater (p < 0.05) when injection was performed at 11 h after insemination compared to 19 h after insemination. Development of non-injected embryos was greater (p < 0.01) than that of injected embryos. There was no difference in transgene detection frequency in embryos of all developmental states between treatments (53% at 11; 50% at 15; 48% at 19 h after insemination). Injected embryos testing positive for the presence of the transgene exhibited increased development over negative embryos (p < 0.01). Greater development efficiencies can be obtained in microinjected bovine embryos when injection is performed early in pronuclear formation.

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The fate of DNA injected into mammalian oocytes and zygotes at different stages of the cell cycle

Cited by 16 publications

References 18 publications

Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression

Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression

Frequency of detection of exogenous DNA 7, 14, and 21 days after microinjection of bovine zygotes

Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos

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