Method for the rapid selection of homozygous primary cell lines for the production of transgenic animals by somatic cell nuclear transfer
First Claim
1. A method for the accelerated production of transgenic animals homozygous for a selected trait comprising:
- transfecting a non-human mammalian cell-line with a given transgene construct containing at least one DNA encoding a desired gene;
selecting a cell line(s) in which the desired gene has been inserted into the genome of that cell or cell-line;
performing a first nuclear transfer procedure to generate a first transgenic animal heterzygous for the desired gene;
characterizing the genetic composition of said first heterzygous transgenic animal;
selecting cells homozygous for the desired transgene through the use of a selective agent;
characterizing surviving cells using known molecular biology methods; and
picking surviving cells or cell colonies cells for use in a second round of nuclear transfer or embryo transfer; and
producing a second transgenic animal homozygous for a desired transgene.
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Abstract
The present invention provides for the production of homozygous primary cells that carry a specific transgenic integration of interest on both chromosomes by bypassing breeding. These cell lines can they be used for the accelerated production of homozygous transgenic animals by somatic cell nuclear transfer. The invention is thus useful in the production of transgenic ungulate animals capable of producing desired biopharmaceuticals in their milk at higher yield than a comparable heterzygote. By combining the selection techniques of the current invention with somatic cell nuclear transfer it can be applied to large animals, where there is a strong need to shorten the time to homozygosity.
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Citations
25 Claims
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1. A method for the accelerated production of transgenic animals homozygous for a selected trait comprising:
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transfecting a non-human mammalian cell-line with a given transgene construct containing at least one DNA encoding a desired gene;
selecting a cell line(s) in which the desired gene has been inserted into the genome of that cell or cell-line;
performing a first nuclear transfer procedure to generate a first transgenic animal heterzygous for the desired gene;
characterizing the genetic composition of said first heterzygous transgenic animal;
selecting cells homozygous for the desired transgene through the use of a selective agent;
characterizing surviving cells using known molecular biology methods; and
picking surviving cells or cell colonies cells for use in a second round of nuclear transfer or embryo transfer; and
producing a second transgenic animal homozygous for a desired transgene. - View Dependent Claims (12, 13, 19, 25)
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2-11. -11. (canceled)
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14-18. -18. (canceled)
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20-24. -24. (canceled)
Specification