Effect of serum concentration, method of trypsinization and fusion/activation utilizing transfected fetal cells to generate transgenic dairy goats by somatic cell nuclear transfer.
This work was performed within a commercial nuclear transfer program to investigate different methods for synchronizing donor cell cycle stage, for harvesting donor cells, and for fusion and activation of reconstructed caprine embryos. Primary fetal cells isolated from day 35 to day 40 fetuses were co-transfected with DNA fragments encoding both the heavy and light immunoglobulin chains of three different monoclonal antibodies and neomycin resistance. Four neomycin resistant cell lines for each antibody were selected, expanded, and aliquots were both cryopreserved for later use as karyoplast donors or used for further genetic characterization. Transfected fetal cells were cultured in 0.5% FBS to synchronize G0/G1 cell cycle stage cells, then re-fed with 10% FBS prior to use to allow donor cells to re-enter the cell cycle. Alternatively, transfected fetal cells were grown to confluence in 10% FBS to induce contact inhibition to synchronize G0/G1 cell cycle stage cells. Adherent monolayers of transfected fetal donor cells were harvested by either partial or complete trypsinization. Donor cells were simultaneously fused and activated with enulceated in vivo produced ovulated oocytes from superovulated does. Half of the fused couplets received an additional electrical activation pulse and non-fused couplets were re-fused. Four live offspring were produced from 587 embryos generated from cell lines cultured in 0.5% FBS, while one live offspring was produced from 315 embryos generated from cell lines cultured in 10% FBS (0.7% versus 0.3% embryos transferred, respectively, P > 0.05). Five offspring were produced from 633 embryos generated from cell lines harvested by partial trypsinization (0.8% embryos transferred), and no offspring were produced from 269 embryos generated from cell lines harvested by complete trypsinization. Four live offspring were produced from 447 embryos generated from re-fused couplets, and one live offspring was produced from 230 embryos generated from fused couplets that received an additional electrical activation pulse (0.9% versus 0.4% embryos transferred, respectively, P > 0.05). These results suggest that low-serum culture of transfected goat fetal cells and harvest by partial trypsinization may be more efficient methods for generating transgenic goats by somatic cell nuclear transfer. In addition, re-fusion of non-fused couplet or an additional activation step was successful for producing live offspring.
Melican
David
D
GTC Biotherapeutics, 300 Charlton Road, Spencer, MA 01562, USA.
Butler
Robin
R
Hawkins
Nathan
N
Chen
Li-How
LH
Hayden
Evelyn
E
Destrempes
Margaret
M
Williams
Jennifer
J
Lewis
Tanya
T
Behboodi
Esmail
E
Ziomek
Carol
C
Meade
Harry
H
Echelard
Yann
Y
Gavin
William
W
D000818:Animals; D030801:Animals, Genetically Modified; D000911:Antibodies, Monoclonal / Q000235:genetics; D001769:Blood; D002453:Cell Cycle; D002459:Cell Fusion; D002478:Cells, Cultured; D015925:Cryopreservation; D003470:Culture Media; D004351:Drug Resistance / Q000235:genetics; D004624:Embryo Transfer; D005260:Female; D005333:Fetus / Q000166:cytology; D006041:Goats / Q000196:embryology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D009355:Neomycin; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000648:ultrastructure; D014162:Transfection; D014357:Trypsin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003470:Culture Media; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D014357:Trypsin; D009355:Neomycin