Method of enriching for mammalian stem cells

• US 6,146,888 A
• Filed: 12/29/1995
• Issued: 11/14/2000
• Est. Priority Date: 04/21/1993
• Status: Expired due to Term

• Current Assignee: University of Edinburgh
• Original Assignee: University of Edinburgh
• Inventors: Mountford, Peter Scott, Smith, Austin Gerard
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): Wilson, Michael C.
• Application Number:
  US08/535,141
• Time in Patent Office: 1,782 Days
• Field of Search: 435/240.2, 435/320.1, 800/2
• US Class Current: 435/325
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/02 Animal zootechnically ameliorated
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 14/70596 Molecules with a "CD"-designation not provided for elsewhere
• CPC Class Codes: C07K 14/81 Protease inhibitors
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/42 being an intron or intervening sequence for splicing and/or stability of RNA
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 2840/203 : having an IRES
• CPC Class Codes: C12N 2840/44 : being a specific part of the splice mechanism, e.g. donor, acceptor
• CPC Class Codes: C12N 5/0623 : Stem cells
• CPC Class Codes: C12N 9/12 : transferring phosphorus containing groups, e.g. kinases (2.7)

Abstract

Mammalian stem cells are obtained and maintained in vitro whose genome has at least one nucleic acid construct encoding an antibiotic resistance gene operatively linked to a promoter specific for mammalian stem cells. The preferential expression of the antibiotic resistance gene in the stem cells results in the preferential survival of the stem cells in the presence of antibiotic.

Claims

1. A method of enriching a population of mammalian cells for stem cells comprising:

a) providing in vitro a mixed population of mammalian cells whose genome comprises at least one nucleic acid construct encoding an antibiotic resistance gene operatively linked to a promoter which preferentially expresses said antibiotic gene in mammalian stem cells,

b) culturing said mixed population of mammalian cells in vitro under conditions conducive to cell survival wherein the preferential expression of said antibiotic resistance gene results in the preferential survival of mammalian stem cells in the presence of antibiotic.

2. The method according to claim 1 wherein the mammalian stem cells are selected from the group consisting of unipotential stem cells and pluripotential stem cells.

3. The method according to claim 1 wherein the antibiotic resistance gene is a neomycin resistance gene.

4. The method according to claim 1 wherein the genome of said mixed population of mammalian cells comprises two nucleic acid constructs each encoding an antibiotic resistance gene operatively linked to a promoter which preferentially expresses the antibiotic resistance gene in mammalian stem cells.

5. The method according to claim 1 wherein expression of the antibiotic resistance gene results from the introduction of a stably integrated, episomally maintained or transiently maintained nucleic acid construct.

6. The method according to claim 1 wherein expression of the antibiotic resistance gene results from operatively inserting the antibiotic resistance gene into an endogenous gene of the mixed population of mammalian cells.

7. The method according to claim 1 wherein the antibiotic resistance gene has been introduced into the mixed population of mammalian cells by transfection, lipofection, injection, ballistic missile, viral vector or electroporation.

8. The method according to claim 1 wherein the mixed population of cells is isolated from the group consisting of a cell line or an animal.

9. The method according to claim 1 wherein the promoter is an Oct4 promoter.

10. The method according to claim 1 wherein the antibiotic resistance gene is a neomycin phosphotransferase gene.

11. The method according to claim 1 wherein the promoter is preferentially expressed in hematopoietic stem cells.

12. The method according to claim 11 wherein the promoter is a CD34 promoter.

13. The method according to claim 1 wherein the mammalian stem cells are selected from the group consisting of embryonic stem cells and somatic stem cells.

14. The method according to claim 1 wherein the mammalian stem cells are selected from the group consisting of hematopoietic stem cells and neural stem cells.

15. An isolated mammalian stem cell comprising at one nucleic acid construct encoding an antibiotic resistance gene operatively linked to a promoter which preferentially expresses said antibiotic gene in said mammalian stem cell.