Carbohydrate determinant selection system for homologous recombination

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.

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 (α1,3GT), A-transferase, or B-transferase.  
     
     
         10 . The method of  claim 3 , wherein the transgene encodes α(1,2)fucosyltransferase (α1,2FT), A-transferase, or B-transferase.  
     
     
         11 . The method of  claim 5 , wherein the endogenous gene encodes α1,3GT, and the transgene encodes α1,2FT.  
     
     
         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.