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.

Description

RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 10 / 850,344, filed May 19, 2004, which is a continuation of U.S. application Ser. No. 09 / 957,659, filed Sep. 20, 2001 (now abandoned), which is a continuation-in-part of U.S. Application Ser. No. 09 / 755,003, filed Jan. 5, 2001 (now U.S. Pat. No. 6,784,336), which claims the benefit of the filing date of U.S. Provisional Application No. 60 / 234,378, filed Sep. 20, 2000 and the filing date of U.S. Provisional Application No. 60 / 255,970, filed Dec. 15, 2000. This application is also related to U.S. application Ser. No. 10 / 787,847, filed Feb. 26, 2004 (now abandoned). The entire teachings of the referenced applications are incorporated herein by reference.GOVERNMENT SUPPORT[0002]Work described herein was supported, in whole or in part, by National Institutes of Health Grants No. 5-R35-CA44339 and RO1-CA84198. The United States government has certain rights in the invention.BACKGROUND OF THE INVENTION...

AI Technical Summary

Benefits of technology

[0005]The present method makes it possible to include multiple mutations or alterations in the same pluripotent cells (e.g., embryonic stem (ES) cells) or cell lines (e.g., ES cell lines) before producing an animal from the ES cells or ES cell lines. The present invention relates to methods of producing non-human mammals that, thus, avoid the time-consuming step of breeding chimera to produce the desired offspring. As is evident from the work described herein, mutant or targeted offspring, particularly mice, that are entirely derived from ES cells or ES cell lines and survive postnatally have been produced without the need to produce chimeric intermediates. Mutations introduced into the non-inbred pluripotent cells or cell lines can be non-random or targeted alterations or can be random or non-targeted alterations. The products of either approach are referred to herein as mutant. In those embodiments in which mutations are non-random or targeted, the resulting products can also be referred to as targeted (e.g., targeted ES cells, targeted ES cell lines, targeted non-human mutant mammals, such as targeted mutant mice). Alterations can be of a variety of types, including deletion, addition, substitution, or modification of all or a portion of DNA (e.g., a gene, regulatory element) in the ES cells. These alterations include addition of a gene or gene portion not normally present in the ES cells or ES cell lines. Non-mutant mice that are derived entirely from ES cells or ES cell lines and survive postnatally can also be produced using the method described. The present methods of producing mice, particularly mutant mice, make it possible to produce offspring, particularly mutant offspring, very efficiently, particularly in comparison with other methods.

[0006]The present invention also relates to a method for deriving fertile XO female mice from non-inbred (F1) mouse male ES cells or non-inbred (F1) mouse male cell lines and a method of deriving males and females carrying all genetic alterations introduced into a single non-inbred ES clone, such as a targeted non-inbred mouse ES cell clone. Breeding of the mutant males and females allows the production of a mutant mouse strain derived from a single non-inbred ES cell clone, such as a targeted (e.g., multiply targeted ES cell clone), without outcrossing the mutant animal with a wildtype partner, as is required in presently available methods.

Problems solved by technology

Producing desired genetically manipulated mammals, even those for which the breeding cycle is relatively brief, requires considerable time, as well as resources, using current methods.