Nuclear Donor Cell selection:

• fibroblasts
• cumulus cells
• sertoli leydig cells
• Primitive Pluripotent Stem cells????
Established criteria:
• quiescent population G0 or G1 Phase
• serum starvation vs contact inhibition
• Telemerase expression?
• Donor Cell Culture
• Ideal atmospheric conditions?
• Culture media
• Additional additives:
• Pantothenic Acid
• Carnosine???
• Creatine???
• L-Carnitine???
• Various antioxidants: consider synthetic free radical inhibitors
Oocyte Maturation
• All-Trans Retinoic Acid
Nuclear Transfer
• Elecrofusion
• Direct Nuclear Transfer ( Honolulu Technique)
Blastocyst Culture

Oocyte synchronization

• chemical agents

transfer methodology
• electroportation
• direct nuclear transfer
Nuclear Reprogramming
• demethylation/remethylation
• time delay between transfer and activation
Activation Methods
• electrical
• chemical
• Nitric Oxide
Species considerations
• onset point of nuclear transcription
• endogenous antioxidant systems
Epigenetic Factors

Cell Cycle Synchonization

• Serum Starvation
• contact inhibition
Culture systems
• endometrial coculture
• three phase chemical media
• coculture with other cell types?: cumulus cell