Method of producing mutant mice

Details Method of producing mutant mice Method of producing mutant mice

2001-01-05

2004-08-31

Falk, Anne-Marie (Department: 1636)

Multicellular living organisms and unmodified parts thereof and

Method of making a transgenic nonhuman animal

C800S018000

Reexamination Certificate

active

06784336

ABSTRACT:

BACKGROUND OF THE INVENTION
In the past two decades, considerable effort has been invested in producing non-human mammals, such as mutant or transgenic mammals, such as mice, and during that time, a variety of methods have been developed. In order to produce a desired mutant animal, such as a mouse, using any of the presently available methods, one must first produce chimeras and breed the chimeras to produce homozygous offspring; production of offspring which are not chimeric requires two breeding cycles for each gene. This is the case for each mutation to be introduced and, if offspring exhibiting more than one mutation are desired, additional breeding cycles are required. For example, if mutant mice bearing six different alterations (e.g., six different genes) are to be produced, approximate breeding time will be two years. 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. It would be very valuable if a more efficient method of producing mutant mammals, such as mice, were available.
SUMMARY OF THE INVENTION
The present invention relates to methods of producing non-human mammals, which can be mutant non-human mammals or non-mutant non-human mammals, such as mice, that do not require production of chimera or chimeric offspring (offspring that consist of cells that are derived from more than one zygote). The invention is a method of producing non-human mammals, such as mutant or non-mutant mice, by tetraploid blastocyst complementation using non-inbred pluripotent cells, such as non-inbred ES cells.
The present method makes it possible to include multiple mutations or alterations in the same pluripotent cells (e.g., embryonic stem (ES) cells) before producing an animal from the ES cells. 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 and survive postnatally have been produced without the need to produce chimeric intermediates. Mutations introduced into the non-inbred pluripotent cells 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 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. Non-mutant mice that are derived entirely from ES cells 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.
The present invention also relates to a method for deriving fertile XO female mice from non-inbred (F1) mouse male ES cells 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.
The present invention also relates to non-human mammals, particularly mutant non-human mammals, such as mutant mice, produced by the methods; cells obtained from the mutant or non-mutant non-human mammals and cell lines produced from these cells. A particular embodiment is cells obtained from mutant or non-mutant mice produced by a method of the present invention; cells obtained from the mice and cell lines produced from such cells. The invention further relates to a method of producing blastocysts useful in the method of producing mutant or non-mutant mammals, such as mouse blastocysts (non-mutant or mutant) useful for producing non-mutant or mutant mice by the method described herein and blastocysts produced by the method.
In particular embodiments, mutant non-human mammals (e.g., mutant mice) are produced to mimic or serve as a model for a condition (e.g., a neurological, muscular or respiratory condition, cancer, viral infection, arthritis,) that occurs in another species, such as in humans. They are used to identify new drugs that have a therapeutic or preventive effect on the condition or assess the ability of known drugs to act as therapeutics or preventatives. Thus, the present invention encompasses methods in which the mutant non-human mammals (particularly mutant mice) are used, such as in a method of screening to identify a new drug that inhibits the occurrence of (prevents the onset, reduces the extent or severity of) or reverses a condition caused by or associated with the genetic alteration(s) and a method of screening known drugs for those that inhibit onset of or reverse such conditions. Drugs identified by methods in which the mutant mammals of the present invention are used are also the subject of this invention. These include drugs that inhibit onset of a condition (prevent the onset or reduce the extent to which the condition is established or severity of the condition), referred to as preventatives or prophylactic drugs and drugs that reverse (partially or completely) or reduce the extent or duration of the condition once it has occurred.
DETAILED DESCRIPTION OF THE INVENTION
As described herein, Applicants have demonstrated that genetic background is a crucial parameter controlling postnatal survival of offspring that are entirely derived from ES cells. That is, heterozygosity of the genome of the pluripotent donor cell (e.g., heterozygosity of the donor ES cell genome) is critical for postnatal survival of offspring whose development is achieved without the contribution of normal cells derived from the host embryo. Further, Applicants have demonstrated that non-human mammals, particularly mice, can be generated without the need to first create a chimeric intermediate. The ability to derive offspring (e.g., mice) directly from ES cells without the need to produce chimeric intermediates is a distinct advantage, not only because it avoids the time consuming and expensive step of producing chimera, but also because it facilitates the generation of offspring with multiple genetic alterations. The generation of F1 ES cell-tetraploid mice provides a simple procedure for directly deriving animals with complex genetic alterations without the need to create a chimeric intermediate. The tetraploid technology in combination with the use of F1 cells allows assembly or production of multiple genetic alterations in the same ES cell clone by consecutive gene targeting cycles in vitro. The resulting multiply targeted F1 ES cell clone is introduced into tetraploid blastocysts to produce an embryo that is then transferred to an appropriate foster mother and permitted to develop to term. Thus, a transgenic animal with one or multiple desired or selected genetic alterations can be generated without the need for production of chimeric founders and outbreeding with wild type mice.
Also described herein is a strategy for deriving fertile XO females from F1 male ES cells and a method of breeding a mutant mouse strain derived from a given multiply targeted ES cell clone without outcrossing the mutant animal with a wild type partner. This avoids time consuming and costly outcrossing, which would otherwise be necessary. Because each F1 ES cell line is of a given

Affiliated with

Also associated with

Rating

Method of producing mutant mice does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Method of producing mutant mice, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of producing mutant mice will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3305605