Method of producing non-human mammals

Abstract

A method of producing mutant/targeted non-human mammals, such as mutant mice that does not require production of chimera and permits the introduction of multiple mutations in embryos and, thus, avoids the necessity of breeding to combine all of the desired mutations in a single animal. The method is efficient in producing ES mice.

1 . A method of identifying XO F1 ES cells, comprising screening a population of F1 ES cells for F1 ES cells that are XO F1 ES cells, wherein said population is a mixture of XO F1 ES cells and XY F1 ES cells. 
     
     
         2 . The method of  claim 1 , wherein screening is carried out to identify F1 ES cells in which spontaneous loss of the Y chromosome has occurred, thereby producing XO F1 ES cells. 
     
     
         3 . The method of  claim 2 , wherein said population is a population of wildtype F1 ES cells or a population of mutant F1 ES cells. 
     
     
         4 . The method of  claim 3  wherein the F1 ES cells are mouse cells. 
     
     
         5 . The method of  claim 3 , wherein screening is carried out using a Y chromosome probe against repetitive elements or PCR amplification. 
     
     
         6 . A method of isolating XO F1 ES cells from XY F1 ES cells, comprising (a) screening a population of F1 ES cells for loss of the Y chromosome, wherein said population is a mixture of XO F1 ES cells and XY F1 ES cells; and (b) isolating F1 ES cells lacking the Y chromosome, thereby isolating XO F1 ES cells. 
     
     
         7 . The method of  claim 6 , wherein said population is a population of wildtype F1 ES cells or a population of mutant F1 ES cells. 
     
     
         8 . The method of  claim 7 , wherein the F1 ES cells are mouse cells. 
     
     
         9 . The method of  claim 7 , wherein screening is carried out using a Y chromosome probe against repetitive elements or PCR amplification. 
     
     
         10 . The method of  claim 7 , wherein the F1 ES cells are generated by introducing into male F1 ES cells a negative selection marker under conditions appropriate for insertion of the negative selection marker in the Y chromosome of male F1 ES cells, thereby producing a mixture of F1 ES cells comprising male F1 ES cells in which the negative selection marker is inserted in the Y chromosome and other male F1 ES cells, some of which do not contain a Y chromosome; and subjecting the resulting F1 ES cells to conditions that result in the death of male F1 ES cells in which the Y chromosome has the negative selection marker inserted therein and do not result in the death of male F1 ES cells that lack a Y chromosome. 
     
     
         11 . A method of producing an XO female non-human mammal, comprising introducing XO F1 ES cells identified using the method of claim  49  into tetraploid blastocysts of the same mammalian species under conditions that result in production of an embryo and transferring the resulting embryo into a foster mother which is maintained under conditions that result in development of live offspring, thereby producing an XO female non-human mammal. 
     
     
         12 . The method of  claim 11 , wherein said XO F1 ES cells are wildtype XO F1 ES cells or mutant XO F1 ES cells. 
     
     
         13 . The method of  claim 11 , wherein said XO F1 ES cells are mouse cells and the non-human mammal is a mouse. 
     
     
         14 . A non-human mammal produced by the method of  claim 11 . 
     
     
         15 . A non-human mammal produced by the method of  claim 12 . 
     
     
         16 . A mouse produced by the method of  claim 13 . 
     
     
         17 . A method of producing an XO female mouse, comprising introducing mouse XO F1 cells identified using the method of  claim 1  into tetraploid mouse blastocysts under conditions that result in production of an embryo and transferring the resulting embryo into a foster mother which is maintained under conditions that result in development of live offspring, thereby producing an XO female mouse. 
     
     
         18 . The method of  claim 17 , wherein the mouse XO F1 ES cells are wildtype mouse XO F1 ES cells or mouse mutant XO F1 ES cells. 
     
     
         19 . A mouse produced by the method of  claim 17 . 
     
     
         20 . A mouse produced by the method of  claim 18 . 
     
     
         21 . An XO female mouse produced by (a) introducing mouse XO F1 ES cells into tetraploid mouse blastocysts under conditions that result in production of an embryo, said mouse XO F1 ES cells identified by screening a population of mouse F1 ES cells for F1 ES cells that are XO F1 ES cells, wherein said population is a mixture of mouse XO F1 ES cells and mouse XY F1 ES cells; and (b) transferring the resulting embryo into a foster mother which is maintained under conditions that result in development of live offspring, wherein the live offspring are XO female mice. 
     
     
         22 . The XO female mouse of  claim 21 , wherein screening is carried out to identify F1 ES cells in which spontaneous loss of the Y chromosome has occurred, thereby producing XO F1 ES cells. 
     
     
         23 . The XO female mouse of  claim 22 , wherein said population of mouse F1 ES cells is a population of mouse wildtype F1 ES cells or a population of mouse mutant F1 ES cells. 
     
     
         24 . The XO female mouse of  claim 22 , wherein screening is carried out using a Y chromosome probe against repetitive elements or PCR amplification.