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

Rebecca L. Krisher, John R. Gibbons, Rudolfo S. Canseco, John L. Johnson, Christopher G. Russell, D. R. Notter, William H. Velander, F. C. Gwazdauskas

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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 11h 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 15h 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 19h 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.

Original languageEnglish (US)
Pages (from-to)226-231
Number of pages6
JournalTransgenic Research
Volume3
Issue number4
DOIs
StatePublished - Jul 1 1994

Fingerprint

Microinjections
Insemination
embryo (animal)
Embryonic Structures
insemination
cattle
DNA
Genes
genes
Transgenes
transgenes
Semen Preservation
injection
Morula
pronucleus
Injections
Zygote
Buffaloes
morula
Blastocyst

Keywords

  • DNA integration
  • PCR
  • micromanipulation
  • transgenics

ASJC Scopus subject areas

  • Biotechnology
  • Animal Science and Zoology
  • Agronomy and Crop Science
  • Genetics

Cite this

Krisher, R. L., Gibbons, J. R., Canseco, R. S., Johnson, J. L., Russell, C. G., Notter, D. R., ... Gwazdauskas, F. C. (1994). Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos. Transgenic Research, 3(4), 226-231. https://doi.org/10.1007/BF02336775

Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos. / Krisher, Rebecca L.; Gibbons, John R.; Canseco, Rudolfo S.; Johnson, John L.; Russell, Christopher G.; Notter, D. R.; Velander, William H.; Gwazdauskas, F. C.

In: Transgenic Research, Vol. 3, No. 4, 01.07.1994, p. 226-231.

Research output: Contribution to journalArticle

Krisher, RL, Gibbons, JR, Canseco, RS, Johnson, JL, Russell, CG, Notter, DR, Velander, WH & Gwazdauskas, FC 1994, 'Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos', Transgenic Research, vol. 3, no. 4, pp. 226-231. https://doi.org/10.1007/BF02336775
Krisher RL, Gibbons JR, Canseco RS, Johnson JL, Russell CG, Notter DR et al. Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos. Transgenic Research. 1994 Jul 1;3(4):226-231. https://doi.org/10.1007/BF02336775
Krisher, Rebecca L. ; Gibbons, John R. ; Canseco, Rudolfo S. ; Johnson, John L. ; Russell, Christopher G. ; Notter, D. R. ; Velander, William H. ; Gwazdauskas, F. C. / Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos. In: Transgenic Research. 1994 ; Vol. 3, No. 4. pp. 226-231.
@article{a40df0642ee04a75bd9e1ff9f4e051e2,
title = "Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos",
abstract = "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 11h 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 15h 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 19h 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.",
keywords = "DNA integration, PCR, micromanipulation, transgenics",
author = "Krisher, {Rebecca L.} and Gibbons, {John R.} and Canseco, {Rudolfo S.} and Johnson, {John L.} and Russell, {Christopher G.} and Notter, {D. R.} and Velander, {William H.} and Gwazdauskas, {F. C.}",
year = "1994",
month = "7",
day = "1",
doi = "10.1007/BF02336775",
language = "English (US)",
volume = "3",
pages = "226--231",
journal = "Transgenic Research",
issn = "0962-8819",
publisher = "Springer Netherlands",
number = "4",

}

TY - JOUR

T1 - Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos

AU - Krisher, Rebecca L.

AU - Gibbons, John R.

AU - Canseco, Rudolfo S.

AU - Johnson, John L.

AU - Russell, Christopher G.

AU - Notter, D. R.

AU - Velander, William H.

AU - Gwazdauskas, F. C.

PY - 1994/7/1

Y1 - 1994/7/1

N2 - 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 11h 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 15h 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 19h 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.

AB - 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 11h 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 15h 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 19h 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.

KW - DNA integration

KW - PCR

KW - micromanipulation

KW - transgenics

UR - http://www.scopus.com/inward/record.url?scp=0028465435&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028465435&partnerID=8YFLogxK

U2 - 10.1007/BF02336775

DO - 10.1007/BF02336775

M3 - Article

C2 - 7920738

AN - SCOPUS:0028465435

VL - 3

SP - 226

EP - 231

JO - Transgenic Research

JF - Transgenic Research

SN - 0962-8819

IS - 4

ER -