Carbohydrate determinant selection system for homologous recombination

• US 20060057719A1
• Filed: 09/02/2005
• Published: 03/16/2006
• Est. Priority Date: 03/21/2001
• Status: Abandoned Application

• Current Assignee: Chris Denning, Clark A. John, Schiff J. Michael
• Original Assignee: Chris Denning, Clark A. John, Schiff J. Michael
• Inventors: Clark, A. John, Denning, Chris, Schiff, J. Michael
• Application Number:
  US11/219,419
• Publication Number:
  US 20060057719A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/02 Animal zootechnically ameliorated
• CPC Class Codes: A01K 2267/025 Animal producing cells or organs for transplantation
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: A61K 2035/122 for inducing tolerance or supression of immune responses
• 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 2517/02 Cells from transgenic animals
• CPC Class Codes: C12N 2517/04 Cells produced using nuclear transfer
• CPC Class Codes: C12N 2517/10 Conditioning of cells for in vitro fecondation or nuclear transfer
• CPC Class Codes: C12N 2800/108 episomal vectors
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase

Abstract

This invention provides a system for selecting a cell that has undergone genetic alteration by homologous recombination from amongst a population of cells that do not have the alteration. The successfully targeted cells are identified and separated according to surface glycosylation that has changed as a result of the homologous recombination. The recombination event may inactivate an endogenous gene, or introduce a transgene, either of which encodes a carbohydrate modulating enzyme, such as α(1,3)galactosyltransferase or α(1,2)fucosyltransferase. Altering carbohydrate modulating enzymes can be done for producing tissue with altered carbohydrate determinants, or as a means for tracking inactivation or insertion of other genetic elements for a variety of purposes.

Claims

1. A method for selecting a mammalian cell that has undergone a genetic alteration by homologous recombination from amongst a population of cells that do not have said alteration, comprising separating cells according to a surface carbohydrate determinant that has changed as a result of the homologous recombination.

2. The method of claim 1, wherein the homologous recombination inactivates an endogenous gene in the cell that encodes an enzyme affecting a surface carbohydrate determinant.

3. The method of claim 1, wherein the homologous recombination introduces a transgene into the genome of the cell that encodes an enzyme affecting a surface carbohydrate determinant.

4. The method of claim 1, wherein the homologous recombination introduces a site-specific recombinase recognition sequence into the cell.

5. The method of claim 2, wherein the homologous recombination also introduces a transgene into the genome of the cell that that encodes an enzyme affecting a surface carbohydrate determinant.

6. The method of claim 2, wherein the endogenous gene encodes a glycosyltransferase, but the transgene does not.

7. The method of claim 3, wherein the transgene encodes a glycosyltransferase, but the endogenous gene does not.

8. The method of claim 5, wherein both the endogenous gene and the transgene encode different glycosyltransferases.

9. The method of claim 2, wherein the endogenous gene encodes α

(1,3)galactosyltransferase (α

1,3GT), A-transferase, or B-transferase.

10. The method of claim 3, wherein the transgene encodes α

(1,2)fucosyltransferase (α

1,2FT), A-transferase, or B-transferase.

11. The method of claim 5, wherein the endogenous gene encodes α

1,3GT, and the transgene encodes α

1,2FT.

12. The method of claim 1, further comprising removing the transgene from the cell having the genetic alteration subsequent to separating it from cells without the genetic alteration.

13. The method of claim 1, comprising combining the cell population with antibody and complement, such that the antibody binds to a glycosylation determinant present only on cells without the genetic alteration and thereby opsonizes the cells for complement lysis.

14. The method of claim 1, comprising labeling cells with an antibody specific for a glycosylation determinant present only on cells without the genetic alteration, and removing cells that have bound the lectin or antibody.

15. The method of claim 1, comprising labeling cells with an lectin specific for a glycosylation determinant present only on cells without the genetic alteration, and removing cells that have bound the lectin or antibody.

16. The method of claim 15, wherein the cells are labeled with fluorescently conjugated UEA-1 lectin, Helix pomatia lectin, or IB4 lectin.

17. The method of claim 15, comprising sorting single cells from the population into separate wells of a microtiter plate.

18. The method of claims 17, comprising labeling cells with an antibody or lectin specific for a glycosylation determinant present only on the cell that has the genetic alteration, and collecting the cell that has bound the lectin or antibody.

19. The method of claim 1, wherein the cell population is a population of human pluripotent stem cells.

20. The method of claim 1, wherein the cell population is a population of non-human cells suitable as donors for nuclear transfer into recipient cells of the same species.