Methods comprising apoptosis inhibitors for the generation of transgenic pigs

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Stem Cell Related Patent Number US6436701

Title:Derivation of pluripotential embryonic cell lines from ungulate species
Inventors:Evans, Martin John; Cambridge, United Kingdom
Summary:Described herein is a method of deriving pluripotent embryonic stem cells that are isolated from an ungulate species embryonic blastocyst. The invention relates to a process of selecting and growing blastocysts in a tissue culture growth medium which includes both heat-inactivated new born calf serum and heat-inactivated fetal calf serum, after which the blastocysts are then disaggregated either after spontaneous hatching or after mechanical removal of the zone pellucida. Further disclosed are methods of growing stem cell colonies from the disaggregated cells in a tissue culture growth medium from which stem cell colonies are selected according to morphological characteristics and grown in culture. The cells are described as being round, tightly packed cells with large nuclei in relation to cytoplasm, fairly prominent nucleoli, and able to grow in tightly adherent coloedes, with a central flattening as the colonies enlarge. Claims of the invention include the outer, less flattened cells of a larger colony or all the cells of a smaller colony without central flattening which are described as readily disaggregated by trypsinization into small spherical cells which have a bright phase contrast appearance, and if observed after a short period of incubation at 37? C have been shown to exhibit lobular pseudopodia. Applications in the production of transgenic animals are included.
Abstract:A method of selecting and growing pluripotential embryonic stem cells isolated from an ungulate species blastocysts of embryos that develop by way of an embryonic disc is disclosed. The method comprises growing blastocysts in tissue culture growth medium which includes both heat-inactivated new born calf serum and heat-inactivated fetal calf serum; disaggregating the blastocysts either after spontaneous hatching or after mechanical removal of the zone pellucida; growing stem cell colonies from the disaggregated cells in issue culture growth medium; selecting stem cell colonies by morphological characteristics; and growing the selected stem cells in tissue culture growth medium. The cells are round cells, tightly packed with large nuclei in relation to cytoplasm, and fairly prominent nucleoli. They grow in tightly adherent coloedes and as the colonies get larger the cells tend to flatten out in the center of the colony. The outer, less flattened cells of a larger colony or all the cells of a smaller colony without central flattening are readily disaggregated by trypsinization into small spherical cells which have a bright phase contrast appearance, and if observed after a short time of incubation at 37.degree. C. they show lobular pseudopodia.
US Patent Website:Click Here for Full Text of Patent
Title Number:US6436701
Application Number:US1991000669403
Date Filed:23/04/1991
Date Published:20/08/2002
Assignee:Babraham Institute, Cambridge, United Kingdom


 
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