Production of cloned goats after nuclear transfer using adult somatic cells.
The developmental potential of adult somatic nuclei after nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated in a dwarf breed of goat (BELE: Breed Early Lactate Early). Somatic donor cells were obtained from two different sources: 1) adult granulosa cells (GCs) and 2) fetal fibroblasts. Primary GCs were obtained from follicular aspirants after laparoscopic oocyte pick-up (LOPU) and were cryopreserved immediately. Frozen aliquots of cells were thawed and cultured until confluent and were then cultured in low serum for 4 days before use in NT. Immature oocytes were obtained by LOPU and matured before enucleation and NT. Ninety-one adult GC-derived NT embryos were transferred into eight recipients, four of which were confirmed pregnant (50%) at Day 30 by ultrasound. Fifty-four male fetal fibroblast-derived NT embryos were transferred into six recipients, one of which was confirmed pregnant (17%). All pregnancies were maintained through term. Four recipients delivered seven female kids (three sets of twins) derived from the GC cultures (7.7% of embryos transferred). The other recipient delivered two male kids (3.7% of embryos transferred). Birth weights were within the normal range for dwarf goats. One female twin and one male twin died at birth; the remaining kids appeared healthy and normal. DNA analysis confirmed that the kids were genetically identical to their respective donors. These results demonstrated that adult caprine somatic cells could direct normal development after NT.
Keefer
C L
CL
Nexia Biotechnologies, Inc., Vaudreuil-Dorion, Quebec J7V 8P5, Canada. ckeefer@nexiabiotech.com
Keyston
R
R
Lazaris
A
A
Bhatia
B
B
Begin
I
I
Bilodeau
A S
AS
Zhou
F J
FJ
Kafidi
N
N
Wang
B
B
Baldassarre
H
H
Karatzas
C N
CN
D000818:Animals; D002459:Cell Fusion; D002467:Cell Nucleus / Q000235:genetics; D019976:Cloning, Organism; D004624:Embryo Transfer; D005260:Female; D005307:Fertilization in Vitro; D005347:Fibroblasts; D005838:Genotype; D006041:Goats / Q000235:genetics*; D006107:Granulosa Cells / Q000502:physiology; D010535:Laparoscopy; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000254:growth & development / Q000502:physiology; D018807:Polymorphism, Single-Stranded Conformational; D011247:Pregnancy; D020133:Reverse Transcriptase Polymerase Chain Reaction