Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2001-11-30
2004-09-14
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S014000, C800S021000, C800S022000, C800S025000
Reexamination Certificate
active
06791006
ABSTRACT:
TECHNICAL FIELD
The present invention relates to mammals engineered to have a functional deficiency in the LKB1 gene as well as to a preparation method thereof. The human LKB1 gene is a causative gene of Peutz-Jeghers syndrome, and thus such mammals can be used to develop methods to treat the disease and therapeutic agents therefor.
BACKGROUND
Peutz-Jeghers syndrome (MIM 175200, PJS) is a human genetic disease the major go symptoms of which include polyposis in the digestive tract and pigmental spot formation on mucous membranes and skin. PJS is inherited in an autosomal-dominant fashion. In 1997, Hemminki et al. reported that the causative gene for the disease was mapped on p13.3 region of chromosome 19 based on the linkage analysis of PJS patient families (Hemminki et al., Nat. Genet. 15:87-90, 1997). There exists the novel serine/threonine kinase, LKB1, which was found by the present inventors in this region. Based on the fact, Jenne et al. predicted that this gene is a candidate for the causative gene and carried out mutational analysis of the LKB1 (STK11) gene in PJS patients. Their results showed that all the patients tested had mutations in the LKB1 gene (PCT/JP98-05357; Jenne et al., Nat. Genet. 18:38-43, 1998). In addition, other groups also reported similar results, one after another. More than 60 types of mutations in the LKB1 gene have been found to date in PJS patients (Hemminki et al., Nature 391:184-7, 1998; Nakagawa et al., Hum. Genet. 103:168-72, 1998; Resta et al., Cancer Res. 58:4799-801, 1998; Ylikorkala et al., Hum. Mol. Genet. 8:45-51, 1999).
Further, the present inventors have demonstrated that the product of the LKB1 gene is a kinase having the ability of autophosphorylation, and that missense mutations that have been found in PJS patients result in loss of the kinase activity (Mehenni et al., Am. J. Hum. Genet. 63:1641-50, 1998).
Based on these findings, it has been clarified that functional deficiency of the LKB1 serine/threonine kinase due to gene mutations is the cause of PJS.
Further, epidemiological studies have shown that PJS patients have markedly increased risks for the onset of a variety of cancers compared with healthy normal persons. Thus, it has been suggested that the PJS causative gene should have a tumor suppressor gene like activity. In fact, it has been reported that mutations are found in the LKB1 gene in some sporadic cancers unrelated to PJS. Therefore, it has been elucidated that functional inactivation of the LKB1 gene is related to general sporadic cancers (Dong et al., Cancer Res. 58:3787-90, 1998; Rowan et al., J. Invest. Dermatol. 112:509-11, 1999; Guldberg et al., Oncogene 18:1777-80, 1999). However, specific physiological functions of LKB1 in normal cells as well as the mechanism for polyposis or cancerization induced by its functional inactivation has remained obscure.
SUMMARY
This situation led to the present invention, and the object of the present invention is to provide non-human mammals useful for analyzing LKB1 functions and for developing agents to treat diseases caused by LKB1 mutations. More specifically, the object of the invention is to provide knockout animals, in which the expression of the LKB1 gene is artificially suppressed, as well as to provide methods for preparing the animals. In a preferred embodiment, the present invention provides non-human mammals in which deletion of the endogenous LKB1 is achieved in an inducible manner.
The present inventors created mammal models, in which the LKB1 gene is artificially deleted, or in which the deletion can be induced. Specifically, as shown in the Examples, a mouse LKB1 gene (both the genomic DNA and cDNA) was cloned; a vector for homologous recombination was constructed using the cloned gene; the vector was introduced into mouse embryonic stem cells (ES cells) to obtain recombinant clones; and the clone was introduced back to an individual mouse which then enabled acquisition of mice having mutations in the LKB1 gene. The present inventors used Cre-loxP system (described later) in creating the recombinant mouse, and thus, achieved phase-specific and tissue-specific induction of mutations in the LKB1 gene. According to this method, the inventors overcame the previous problem of potential embryonic lethality caused by the inactivation of the gene of interest. The mammals in accordance with the present invention (and cell lines established thereof) are expected to be useful as tools to study the onset mechanism of a variety of diseases caused by LKB1 gene deficiency, such as PJS and cancers, and furthermore, are highly useful tools in the development of therapeutic methods and agents for these diseases. Thus, these mammals and cells are expected to be used for various purposes.
The present invention relates to non-human mammals, in which the expression of the LKB1 gene can be or is artificially suppressed, as well as methods for creating the same. More specifically, the present invention relates to the following:
(1) a non-human mammal in which the expression of the endogenous LKB1 gene can be artificially suppressed;
(2) the non-human mammal of (1), wherein the suppression of the expression of the endogenous LKB1 gene is induced by deleting at least a part of the gene or the regulatory region thereof;
(3) the non-human mammal of (1) or (2), wherein at least a part of the LKB1 gene or the regulatory region thereof in the genome is inserted between at least a pair of loxP sequences;
(4) the non-human mammal of any of (1) to (3), wherein the mammal is a rodent;
(5) the non-human mammal of (4), wherein the rodent is a mouse;
(6) a non-human mammal wherein the expression of the endogenous gene encoding LKB1 is artificially suppressed;
(7) the non-human mammal of (6), wherein the expression of the endogenous gene encoding LKB1 is suppressed by a defect in at least a part of the gene or the regulatory region thereof;
(8) the non-human mammal of (6) or (7), wherein the mammal is a rodent;
(9) the non-human mammal of (8), wherein the rodent is a mouse;
(10) a non-human mammalian cell wherein the suppression of the expression of the endogenous gene encoding LKB1 can be artificially induced, further wherein the cell can be differentiated or developed into an individual mammal;
(11) the non-human mammalian cell of (10), wherein the suppression of the expression of the endogenous gene encoding LKB1 is induced by deleting at least a part of the gene or the regulatory region thereof;
(12) the non-human mammalian cell of (10) or (11), wherein at least a part of the LKB1 gene or the regulatory region thereof in the genome is inserted between at least a pair of loxP sequences;
(13) the non-human mammalian cell of (12), wherein the cell contains the Cre gene in an expressible manner;
(14) the non-human mammalian cell of any of (10) to (13), wherein the cell is a rodent cell;
(15) the non-human mammalian cell of (14), wherein the cell is a mouse cell;
(16) the non-human mammalian cell of any of (10) to (15), wherein the cell is an embryonic stem cell;
(17) a non-human mammalian cell, wherein the expression of the endogenous gene encoding LKB1 is artificially suppressed, further wherein the cell can be differentiated or developed into an individual mammal;
(18) the non-human mammalian cell of (17), wherein the expression of the endogenous gene encoding LKB1 is suppressed by a defect in at least a part of the gene or the regulatory region thereof;
(19) the non-human mammalian cell of (18), wherein the cell can be obtained by expressing the Cre gene in the non-human mammalian cell described in (12);
(20) the non-human mammalian cell of any of (17) to (19), wherein the cell is a rodent cell;
(21) the non-human mammalian cell of (20), wherein the cell is a mouse cell;
(22) the non-human mammalian cell of any of (17) to (21), wherein the cell is an embryonic stem cell;
(23) a method for creating the non-human mammal described in any of (1) to (5), which comprises the following steps:
(a) introducing the non-human mammalian cell described in (16) into an embryo obtained from a pregnant fema
Jenne Dieter E.
Jishage Kou-Ichi
Nezu Jun-Ichi
Ose Asuka
Bertoglio Valarie
Chugai Seiyaku Kabushiki Kaisha
Crouch Deborah
Fish & Richardson P.C.
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