In vitro development of zygotes from prepubertal gilts after microinjection of DNA

Williams, Barry L.; Canseco, R. S.; Knight, James; Johnson, John L.; Velander, William H.; Page, Raymond; Drohan, William N.; Young, Janet M.; Pearson, R.E.; Wilkins, Tracy D.; Gwazdauskas, F.C.

doi:10.2527/1992.7072207x

Cited by 15 publications

(6 citation statements)

References 12 publications

(9 reference statements)

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“…This result is in agreement with early reports for mice (16), pigs (32), and cattle (18,19). These studies suggest that microinjection may reduce initial cleavage rates by damaging the pronucleus of the zygote (24).…”

Section: Discussionsupporting

confidence: 83%

Bovine Follicular Dynamics, Oocyte Recovery, and Development of Oocytes Microinjected with a Green Fluorescent Protein Construct

Chauhan¹,

Nadir²,

Bailey³

et al. 1999

Journal of Dairy Science

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The present study was carried out to 1 ) evaluate the viability of in vitro fertilized zygotes after microinjection of DNA, 2 ) assess the influence of oocyte quality upon the development rate of embryos when injected with DNA, and 3 ) determine the integration frequency of green fluorescent protein DNA into microinjected embryos. Oocytes were aspirated from ovaries of nine nonlactating Holsteins and were categorized into grades A, B, C, and D. At 16 h after in vitro fertilization, approximately half of the pronuclear stage presumptive zygotes were classified as having 1 pronucleus or 2 pronuclei, and they were microinjected with DNA constructs. A potential predictor of DNA integration frequency at d 10 was assessment of the incidence of green fluorescing embryos. The proportion of cleaved embryos that developed to morulae or blastocysts was not different between groups with 1 pronucleus injected (45%), 1 pronucleus uninjected (64%), or 2 pronuclei injected (49%). However, the development of morulae or blastocysts was higher in the group with 2 pronuclei uninjected (69%). The overall developmental score of green fluorescent protein-positive embryos was higher for grade A oocytes (1.3 ± 0.1) than for grade B (0.8 ± 0.1), C (0.6 ± 0.1), or D (0.3 ± 0.1) oocytes. The results show that production of transgenic bovine blastocysts can occur from the microinjection of a presumptive zygote having only one visible pronucleus. Initial oocyte quality is an important factor in selection of oocytes suitable for microinjection of DNA and for preimplantation development to produce bovine transgenic embryos. ( Key words: bovine oocytes, in vitro fertilization, ovum pickup, green fluorescent protein) Abbreviation key: CMV-EGFP = cytomegalovirus enhanced green fluorescent protein, GFP = green fluorescent protein, IVF = in vitro fertilization, IVM = in vitro maturation, PCR = polymerase chain reaction, 0PN = no pronucleus, 1PN = 1 pronucleus, 2PN = 2 pronuclei, WAP6FIX = whey acidic protein factor IX.

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Section: Discussionsupporting

confidence: 83%

Bovine Follicular Dynamics, Oocyte Recovery, and Development of Oocytes Microinjected with a Green Fluorescent Protein Construct

Chauhan¹,

Nadir²,

Bailey³

et al. 1999

Journal of Dairy Science

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“…The DNA injected at late pronuclear stages may be available for integration during this second replication event. Williams et al (1992) demonstrated that the decrease in developmental efficiencies of DNA-injected embryos was due to the DNA itself, not the injection procedure. The extent of the ability of the embryo to repair damage from micromanipulation at different pronuclear stages is unknown.…”

Section: Introductionmentioning

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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“…Oviducts were exteriorized by midventral laparotomy. A retrograde oviductal flush was performed from the utero-tubal junction to the infundibulum with 20 mL of Beltsville Embryo Culture Medium (V. G. Pursel, personal communication), a HEPES-buffered medium for handling embryos in air (Williams et al, 1992b).…”

Section: Methodsmentioning

confidence: 99%

“…1994. 72:1299-1305 A culture system able to support adequate development of porcine embryos microinjected with DNA to the blastocyst stage needs to be refined (Williams et al, 1992b). Among the best development rates for noninjected embryos reported thus far, Krisher et al (1989) found close t o 80% development to the morula or blastocyst stage for one-cell porcine embryos cultured in mouse oviductal explants.…”

Section: Introductionmentioning

confidence: 99%

Effect of culture conditions, donor age, and injection site on in vitro development of DNA microinjected porcine zygotes1

Hajdu

Knight

Canseco³

et al. 1994

Journal of Animal Science

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A series of experiments evaluated development of porcine zygotes microinjected with DNA in three culture media and two incubation temperatures, from postpubertal and prepubertal donors, and between zygotes injected with DNA into the pronucleus and the cytoplasm. Zygotes recovered from 36 postpubertal gilts in Exp. 1 were injected and cultured in modified NCSU-23, modified NCSU-37, and CZB media at 37 degrees C or 39 degrees C for 7 d. In Exp. 2, zygotes were collected from postpubertal or prepubertal gilts, microinjected with DNA, and cultured in modified NCSU-23. In Exp. 3 superovulated prepubertal gilts had DNA injected into the cytoplasm or pronucleus of zygotes. Mean percentages developing to the expanded or hatched blastocyst stage in modified NCSU-23 (42.9) and modified NCSU-37 (40.1) did not differ, but development was greater than that for zygotes cultured in CZB (8.8; P < .05). Development was greater at 39 degrees C (P < .05) than at 37 degrees C (36.5 vs 24.6%). Microinjection of DNA decreased development (P < .05) from that of noninjected controls (18.1 vs 43.1%). Zygotes from postpubertal gilts had a higher percentage (68.0) of expanded and hatched blastocysts than zygotes from prepubertal donors (29.0; P < .05). No development difference was found between DNA injection into the pronucleus (23.1%) or cytoplasm (17.4%), but development was less than for control embryos (64.9%; P < .05). DNA microinjected porcine zygotes can be successfully cultured to the expanded blastocyst stage in modified NCSU-23 and modified NCSU-37 media at 39 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

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In vitro development of zygotes from prepubertal gilts after microinjection of DNA

Cited by 15 publications

References 12 publications

Bovine Follicular Dynamics, Oocyte Recovery, and Development of Oocytes Microinjected with a Green Fluorescent Protein Construct

Bovine Follicular Dynamics, Oocyte Recovery, and Development of Oocytes Microinjected with a Green Fluorescent Protein Construct

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

Effect of culture conditions, donor age, and injection site on in vitro development of DNA microinjected porcine zygotes1

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