|
KMID : 0900919970210020167
|
|
Korean journal of Animal Reproduction
1997 Volume.21 No. 2 p.167 ~ p.176
|
|
|
Preselection of Bovine Blastocysts Expressing Exogeneous Gene Following Microinjection
|
|
|
|
|
Abstract
|
|
|
|
This study was carried out to evaluate the potential for preselection of transgenic embryos prior to transfer into recipient animals. In these experiments, I used a 3.2 kb transgene which contained the neomycin resistance gene (neo) and lac Z gene driven by the beta actin promoter (iacZ Ineo). Oocytes were aspirated from abattoir ovaries, matured in TCM-199 supplemented with 10% fetal bovine serum (FBS), 5 {mu}textrm{g}/ml LH, 0.5 {mu}textrm{g}/ml FSH, 100 unit/ml penicillin, and 100 {mu}textrm{g}/ml streptomycin for 22 to 24 hrs then inseminated with a sperm suspension of 1 X 10^6 sperm/ml containing 5 {mu}textrm{g}/ml of heparin. At 18-20 hrs after insemination, cumulus cells were removed by vortexing and pronuclei of centrifuged zygotes microinjected with the lacZ/neo construct (3 ng/mul). All cultures were carried out in CR1aa with transfected BRL monolayers containing 3 mg/ml BSA, 20 mu/ml NEM amino acids and 40 muI/ml BME amino acids. To identify the appropriate concentration of G418 for selection, non-microinjected zygotes were cultured in the presence of 0, 50, 100 and 200 mug/ml of G418. After 8 days of culture in these treatments, 44/145 (30.3%), 13/150 (8. 7%), 1/151 (0.7%) and 0/134 (0.0%) devel-oped to the blastocyst stage in 0, 50, 100 and 200 mug/ml of G418, respectively. A total of 1,127 zygotes were microinjected and placed into culture (without G418) and subsequently 710 (63.0%) cleaved. At 48 hrs post-injection, embryos (geq2-cell) were randomly assigned to control (medium alone) or G418 (100 {mu}textrm{g}/ml) treatments. A control culture of 740 non-microinjected embryos from the same replicates of embryos were also placed into control medium. After 8 days in culture, 54/343 (15.7%) and 22/367 (6.0 %) of the microinjected embryos developed to the blastocyst stage in control and G418 media, respectively. A total of 151/740 (27.2%) of the non-microinjected embryos placed in the control medium developed to the blastocyst stage. The blastocysts in the control treatment had a mean of 70.7 {pm} 4.7 cells of which 23.1 {pm} 2.6 (32.7%) stained for beta-Gal activity. B1astocysts in the G418 treatment had a mean of 48.8{pm}7.5 cells of which 40.3 {pm} 4.1 (82.6%) stained for beta-Gal ac tivity (P<0.05). In the control treatment 26 of 30 (87.0%) blastocysts had some cells with beta-Gal activity while all of the blastocysts in the G418 treatment had some cell with beta-Gal ac tivity (P<0.05). However, ICM colonies in either control or G418 treatments were not expressed any epiblast cell except of trophetoderm celIs. The beta-actin promoter/lacZ gene may not be e expression or silence expression in epiblast cells These results clearly show an enrichment of blastocysts expressing the transgene in the majority of their cells after culture in the presence of G418. The exogeneous gene was not express a and silence in ICM colonies especiallly epiblast cells except of trophectederm cells. Even though the higher rate cell number expressed of exogeneous gene in the G418 treatments, a total cell number was decrease significantly (P<0.05) of which might be also drop of the establishment of ES like-cell colonies and production of transgenic animals. However, futher studies need to determine the viability of these selected embryos and the avability of production of transgenic offspring.
|
|
|
KEYWORD
|
|
|
beta-actin/neo gene, Microinjection, G418 preselecton, Bovine IVMFC
|
|
|
FullTexts / Linksout information
|
|
|
|
|
Listed journal information
|
|
|
|
|