Insulin, which is secreted by pancreatic .beta.-cells, is a hormone that lowers blood glucose levels, and glucose is one of the important physiological stimulants of its secretion. The mechanisms of glucose-induced insulin secretion have been studied vigorously, primarily based on the glucose metabolism hypothesis, for over the last two decades, [Malaisse W. J., et al., Metabolism, 28:373-386 (1979)]. The hypothesis, which has widely attracted attention, has been placing emphasis on the probability of glucose metabolites in pancreatic .beta.-cells acting as signals for insulin secretion. Among a variety of metabolites, ATP has been thought to be one of the most important molecules, functioning, as a metabolic energy carrier, as the intracellular signal for insulin secretion in pancreatic .beta.-cells [Ashcroft, S. J. H., Biochem. J., 132:223-231 (1973)].
Pancreatic .beta.-cells are electrophysiologically excitable cells, and it was known that the membrane potential of .beta.-cells is the key factor in the stimulus-secretion coupling in insulin release [Henquin J. C., Experimentia 40:1043-1052 (1984)]. It was also known that glucose regulates .beta.-cell membrane potential by controlling the permeability of the membrane to potassium ion [Sehlin, J., Diabetes, 32:820-323 (1983)].
Then, in 1984, after Cook et al. discovered ATP-sensitive potassium channels (K.sub.ATP), a molecule was first revealed that links glucose metabolism with potassium permeability of the membrane [Cook, D. L. et al., Nature, 311:271-273 (1984)]. K.sub.ATP channels, originally discovered in cardiac muscle, were characterized by inhibition of the channels when intracellular ATP concentration is increased [Noma, A., Nature, 305:147-148 (1983)]. Subsequently, K.sub.ATP channels were found in a variety of tissues and cells, including the brain, pituitary gland and skeletal muscles [Ashcroft, FM, Annu. Rev. Neurosci., 11:97-118 (1988)].
According to the insulin secretion mechanism of pancreatic .beta.-cells, ATP produced by glucose metabolism causes closure of the K.sub.ATP channels, which in turn leads to depolarization of the .beta.-cell membrane, which is followed by opening of the voltage-dependent calcium channels, thereby allowing Ca.sup.2+ influx into the .beta.-cells. The thus resulted rise of intracellular Ca.sup.2+ concentration triggers Ca.sup.2+ -dependent insulin secretion [Wollheim, C. B. et al., Physiol. Rev., 61:914-973 (1981)]. Thus, the K.sub.ATP channels of the .beta.-cells, which link the metabolic status of the cell to its membrane potential, are key molecules, and they are thought to act as intracellular ATP- and ADP-sensors, thereby regulating the excitability of the membrane.
Sulfonylureas (Su agents), insulin secretagogue oral hypoglycemic agents which are widely used threapeutics in the treatment of non-insulin dependent diabetes mellitus (NIDDM), were shown to inhibit the activity of K.sub.ATP channels of .beta.-cells [Sturges, N. C. et al., Lancet, 8453:474-475 (1985), De Weille, J., Proc. Natl. Acad. Sci. USA, 85:1312-1316 (1988)]. Electrophysiological studies on K.sub.ATP channels have shown that their kinetics and pharmacological properties vary among tissues, suggesting that K.sub.ATP differs among tissues structurally and functionally [Terzic, A. et al., Am. I. Physiol., 269:C525-C545 (1995)]. Since K.sub.ATP channels in pancreatic .beta.-cells and insulin-secreting cell lines have the property of inward rectification, they could be structurally related to other channels that constitute the inwardly rectifying K.sup.+ channel family. Inwardly rectifying K.sup.+ channels are distinguished from voltage-gated K.sup.+ channels (Kv) in that the former are not activated by membrane depolarization and they allow greater K.sup.+ influx than efflux.
In understanding the structure and function of the inwardly rectifying K.sup.+ channels, a breakthrough was brought about by expression cloning of cDNAs encoding three distinct inwardly rectifying K.sup.+ channels: ROMK1 (Kir 1.1) from rat kidney [Ho, K. et al., Nature, 362:31-38 (1993)], IRK1 (Kir 2.1) from a mouse macrophage cell line [Kubo, Y. et al., Nature, 362:127-133 (1993)], and KGA (Kir 3.1) from rat heart [Dascal, N. et al., Proc. Natl. Acad. Sci. USA, 90:10235-10239 (1993)]. These studies have established new Kir family genes encoding proteins with different structural and functional characteristics from those of Kv super family. Today, subfamilies of Kir are designated Kir 1.0-7.0 according to the nomenclature of Chandy and Gutman [Chandy, K. G., Gutman, G. A., Trends Pharmacol. Sci., 14:434 (1993). Krapivinsky, G. et al., Neuron 20, 995-1005(1998)].
Using a GIRK1 cDNA as a probe, a new member of the Kir family, uK.sub.ATP -1 (Kir 6.1), has been isolated from a variety of tissues [Inagaki, N., J. Biol. Chem., 270:5691-5694 (1995)]. And, a novel Kir gene, human Kir6.2 (referred also to as "BIR"), has been isolated by screening a human genomic library using a Kir 6.1 cDNA as a probe, and, further, mouse Kir6.2 (mBIR) gene has been obtained by screening 7.times.10.sup.5 plaques from a mouse insulin secreting cell line MIN6 cDNA library under standard hybridization conditions, the DNA sequences of which, for human and mouse, respectively, have been disclosed (Japanese Unexamined Patent Publication 77796/97). The DNA sequence of mouse Kir6.2 gene is set forth in Sequence Listing (SEQ ID No:1).
The Kir6.2 is made up of 390 amino acid residues and there is observed 96% identity between human Kir6.2 and mouse Kir6.2. Further, sulfonylurea receptor (SUR) has been found by Aguilar-Bryan et al. in the process of cloning Kir6.2 (BIR) [Aguilar-Bryan, L. et al., Science, 268:423-426 (1995)]. Now it is known that the pancreatic .beta.-cell K.sub.ATP channel comprises at least two subunits, a Kir6.2 (BIR) and a sulfonylurea receptor (SUR), and mutation occurred in the K.sub.ATP channel gene is considered to be one of the main causal factors of non-insulin dependent diabetes mellitus. For the diversity and functions of inward-rectifying potassium channels, see Horio, Y., SEIKAGAKU, 70(2), p.73-83 (1998), and Seino, S. et al., DIABETES REVIEWS,4(2),177-190 (1996).
Today, diabetes mellitus is a disease of a national scale, the number of its patients including potential ones reaching several million in Japan and the United States, and 5-10% of the middle aged or over are estimated to be diabetic patients. Diabetes mellitus is classified into type I (IDDM), which is insulin-dependent, and type II (NIDDM), non-insulin dependent, and the non-insulin dependent type II constitutes more than 90% of the number of the diabetic patients. As aforementioned, the relation between non-insulin dependent diabetes mellitus and ATP-sensitive potassium channels is being gradually elucidated. However, development of new therapeutics for NIDDM has been retarded, for such experimental animals have not been obtained that are necessary for enabling studies on the physiological role of the ATP-sensitive potassium channel and for enabling thereupon development of improved therapeutics for non-insulin dependent diabetes mellitus.
On the other hand, creation of model animals which have mutation in a particular gene is an important means in studying the functions of the gene which causes various pathological states, and leading to development of diagnostic drugs or therapeutics or methods of treatment. Recent advancement in molecular biology has established techniques to introduce deletion or mutation into a desired gene on the mouse chromosomes by means of gene-targeting in mouse embryonic stem cells (ES cells), thereby enabling to produce knockout mice, i.e. a mouse in which a particular gene is destroyed [Capecchi, M. R., Science, 244:1288-1292 (1989)].
The K.sub.ATP channel in pancreatic .beta.-cells comprises subunits, a sulfonylurea receptor (SUR1) and a Kir6.2 [Inagaki, N. et al., Science, 270:1166-1170 (1995), Sakura, H. et al., FEBS Lett., 377:338-344 (1995)], whereas the K.sub.ATP channel in skeletal muscle or cardiac muscle comprises a SUR2A and a Kir6.2 [Inagaki, N. et al., Neuron, 16:1011-1017 (1996)]. As it is thought to be the Kir6.2 that serves to form a K.sup.+ selective pore of the K.sub.ATP channel, we assumed that mice devoid of K.sub.ATP channels could be created by disrupting Kir6.2 gene. Based upon this assumption, the objective of the present invention is to produce gene-defective (knockout) animals, especially gene-knockout mice, in which Kir6.2 gene, the gene of one of the subunits of K.sub.ATP channels, is lost. Such animals are necessary for elucidating the function and physiological role of K.sub.ATP channels, the essential molecules in insulin secretion by pancreatic .beta.-cells, and for development of therapeutics for non-insulin dependent diabetes mellitus.