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,1
,
* Department of Human Genome Technology, Kausa DNA Research Institute;
Laboratory of Pharmacogenomics, Graduate School of Pharmaceutical Sciences, Chiba University, Kisarazu, Chiba, Japan;
Laboratory for Developmental Genetics, Research Center for Allergy and Immunology, Institute of Physical and Chemical Research (RIKEN), Yokohama, Kanagawa, Japan; and
Laboratory for Immunogenomics, Research Center for Allergy and Immunology, Institute of Physical and Chemical Research (RIKEN), Yokohama, Kanagawa, Japan
1Correspondence: 2–6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818 Japan. E-mail: nmanabu{at}kazusa.or.jp
ABSTRACT
Given that thousands of genes exist in the mammalian genome, criteria are needed to prioritize their functional analysis and to decrease the likelihood of producing gene-targeted mice that lack overt phenotypes. Initial analysis efforts are likely to be fruitful if focused on genes encoding large proteins, since at least some large proteins serve as frameworks for intricate assembly of protein complexes, and their inactivation would render definitive, observable phenotypes. Here, we describe the functional characterization of the murine homologues of five human KIAA genes (KIAA1409, KIAA1440, KIAA1447, KIAA1768, and KIAA1276) that encode large proteins. Gene-targeted mice had phenotypic and developmental defects resulting from the functional deletion of three of these five genes. Mice with targeted disruption of KIAA1409 lacked the ability to drink, and those with targeted disruption of KIAA1447 displayed hind leg motor dysfunction. Disruption of KIAA1440 led to embryonic lethality at the blastocyst stage. The high success rate of our approach demonstrates the rationale for the genome-wide functional examination of large proteins in mice using reverse genetics.—Nakayama, M., Iida, M., Koseki, H., Ohara, O. A gene-targeting approach for functional characterization of KIAA genes encoding extremely large proteins.
Key Words: comprehensive analysis • functional genomics • human genome project • KO mice
ALTHOUGH THE HUMAN genome project has reached an important milestone and approaches completion (1
, 2)
, the systematic determination of human gene function remains challenging. One established and powerful method for analyzing gene function in mammals at the organismal level is gene targeting in mice (3)
. However, there are two inevitable problems with this method when applied to the analysis of large genomes. First, given that thousands of genes exist in the typical mammalian genome, criteria are needed to prioritize their functional analysis. Second, because constructing gene-targeted mice is costly and labor intensive, it is imperative to decrease the likelihood of producing mice that lack overt phenotypes. We propose that initial analysis should be focused on genes encoding large proteins, since at least some large proteins serve as frameworks for the intricate assembly of protein complexes, and inactivation of their genes is likely to render definitive, observable phenotypes.
Two-hybrid screening and tandem affinity purification (TAP) have revealed that, in vivo, many proteins function in multiprotein complexes in which large proteins play an integral role, such as the formation of scaffolds for complex assembly and centers for protein-protein interactions (4
5
6
7)
. Indeed, the mutation or disruption of large proteins containing multiple domains has been linked to familial diseases (8)
, underscoring the importance of studying the function of these proteins. Because of their size, however, extraordinarily large proteins are often technically challenging to analyze; thus, comprehensive studies of large proteins tend to be avoided and their cDNAs are often overlooked by conventional methods. As part of an ongoing project aimed at understanding the human genome, we have accumulated over 2000, previously unidentified, human cDNAs that encode exceptionally large proteins (9
10
11
12
13
14
15
16)
. This has been accomplished by using our special long cDNA-enriched libraries and an approach based on random sampling of long cDNA clones that produces large proteins in an in vitro transcription/translation system. These sequences, termed KIAA plus a four digit number, encode proteins that contain a very large number of binding domains, as predicted by in silico analyses [see human unidentified gene-encoded large protein (HUGE) database at http://www.kasusa.or.jp/huge; ref 16
] and by yeast-two hybrid screening (7
; protein-protein interaction database, HUGEppi, and data not shown). The advent of reverse genetics has opened the door to assessing the function of these large genes in transgenic mammalian model systems.
MATERIALS AND METHODS
Criteria for choosing KIAA genes for study in gene-targeted mice
We selected KIAA genes from the HUGE database, which currently contains over 2000 KIAA genes (16)
. This database also contains comprehensive information about the characteristics of certain KIAA genes and their proteins, such as similarities with other genes and proteins already catalogued in other public domain databases [e.g., the Protein Family (Pfam) database] and data from Northern blot analyses, RT-PCR, RT-ELISA, and RH mapping. We selected five KIAA genes using criteria that increase the probability that gene disruption would be manifested phenotypically. These criteria are as follows (1)
. 1) Size: The gene had to encode a protein containing >1000 amino acids. Our previous findings suggest that extremely large proteins have multiple protein binding sites and play central roles in protein complexes vital to normal cell function (7)
. Thus, disruption of these integral proteins would undoubtedly result in observable phenotypic effects (2)
. 2) Limited homology with known proteins: The gene had to encode a protein having <35% homology with proteins currently listed in public domain databases. We avoided choosing genes that encode members of the same protein families, as well as certain species-specific paralogues, since they can contribute to gene compensation leading to lack of phenotype. This was accomplished through an exhaustive amino acid sequence comparison with conserved protein domains catalogued in the Pfam database (17)
. We excluded proteins having motifs with expectation values of <1, which indicate high homology with known protein domains. Because the function of selected proteins by our criteria cannot be predicted in silico, experimental analysis of such proteins should be performed with higher priority (3)
. 3) Region-specific expression: The gene had to be expressed specifically in the adult human brain. We chose the brain because neural deficits are readily manifested phenotypically. Candidate genes were identified via RT-ELISA.
GenBank/EMBL/DDBJ accession numbers
cDNAs of the mouse orthologues KIAA1409, KIAA1440, KIAA1447, KIAA1276, and KIAA1768 were deposited into GenBank/EMBL/DDBJ under the following accession numbers: AB257853, AB257854, AB257855, AB257856, and AB257857, respectively. The sequences of corresponding genomic DNA fragments (Fig. 1
) were also deposited accordingly (AB257858, AB257859, AB257860, AB257861, and AB257862).
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Rapid screening of candidate targeting vectors and construction of targeting vectors
To efficiently screen candidate target vectors to be used for homologous recombination, we constructed a BAC library containing 
40,000 clones derived from the chromosomal DNA of R1 ES cells harvested from the F1 offspring of 129X1/SvJ and 129S1/Sv mice (18)
. These are the same variety of R1 ES cells used to produce our knockout mice. The average size of each BAC clone was 100–150 kbp. The clone sequences were aligned and plated onto 384-well plates. BAC clones were screened by a polymerase chain reaction (PCR)-based screening procedure using an aligned BAC library comprised of suitable length genomic DNAs (10–15 kbp) subcloned into pBluescript SK(-) vectors (Stratagene). These genomic DNAs were identified by DNA sequencing. The targeting vectors were constructed by using a pMC1neoPolyA (Stratagene) plasmid containing an additional poly(A) site as the backbone vector.
Mice
Chimeric mice were created by injecting 3, 2, 1, 1, and 2 independent targeted ES cell clones of KIAA1276, KIAA1409, KIAA1440, KIAA1447, and KIAA1768, respectively, into C57BL/6J blastocysts. The blastocysts were implanted into pseudopregnant C57BL/6J females according to standard procedure. The resulting chimeric mice were crossed to C57BL/6J wild-type (WT) mice, and F2-F4 offspring were used for experiments. To obtain mice of pure genetic background, mice were repeatedly backcrossed with the inbred C57BL/6J strain. After five or six backcrosses (N5 or N6), we confirmed the phenotype of each gene-targeted mouse except that KIAA1447 gene-targeted mice exhibited early embryonic lethality.
Supplementary data
Supplementary data are available at The FASEB Journal Online.
RESULTS
Comprehensive gene analyses using gene-targeted mice requires a systematic, high-throughput approach
Because constructing gene-targeted mice is time-consuming, laborious, and costly, we have implemented an economical and systematic approach for constructing our five KIAA gene-targeted mice. First, the human KIAA genes analyzed in the present study were selected according to three criteria, size, protein homology, and tissue-specific expression, that would increase the probability that their disruption would produce an observable, measurable phenotype. Thus, we chose genes encoding proteins with 1000 amino acids or more, having <35% shared homology with known proteins, encoding proteins with low expectation values (to avoid compensational effects), and those being expressed primarily in the brain (neural defects are readily manifested phenotypically). We especially focus on the function of proteins having no homology with known proteins and few known motifs. Because the function of such proteins cannot be predicted in silico, we strongly believe that experimental analysis of such proteins should be performed with higher priority to have overall knowledge about the organism. We identified murine homologues of these KIAA genes in silico through exhaustive basic local alignment search tool (BLAST) comparison analyses of known mouse expressed sequence tag (EST) databases, and used these sequences as primers for the PCR-based production of murine homologues. A detailed description of the sequence analyses of these homologues is provided in Table 1
.
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Second, after identifying the target genes, we sought a rapid means to screen BAC clones containing genomic DNA fragments of mouse homologue KIAA genes and to increase the probability of their homologous recombination into the genome of mouse embryonic stem (ES) cells. To ensure complete inactivation, we deleted the exon(s) located closest to the N-terminal end of the encoded protein (757 bp deleted in mKIAA1276, 964 bp in mKIAA1409, 606 bp in mKIAA1440, 631 bp in mKIAA1447, and 700 bp in mKIAA1768). To verify the deletions, we isolated the full-length mouse cDNAs incorporated into targeting vectors, aligned them with corresponding mouse genome sequences, and mapped the positions of the deleted exons (Fig. 1)
. Efficient screening of BAC clones containing mouse homologue KIAA genes was carried out using a PCR-based BAC library screening system that consisted of 40,000 clones, the aligned sequences of which were plated onto 384-well plates. The clones were derived from chromosomal DNA extracted from R1 ES cells, the same cell type used for homologous recombination and implantation into pseudopregnant female mice. We made our vectors using the identical genomic DNA of recipient R1 ES cells, because the frequency of homologous recombinant increases when the targeting vector is constructed with DNA derived from recipient ES cells (19)
. We attained a high frequency of homologous recombination: 1 in 9, 1 in 4, 1 in 7, 1 in 9, and 1 in 27 targeting vectors containing knockouts of KIAA1409 (20)
, KIAA1440 (20)
, KIAA1447 (21)
, KIAA1768 (22)
, and KIAA1276 (23)
, respectively, successfully recombined.
Molecular biological information to predict the molecular mechanism underlying phenotypes of KIAA gene-targeted mice
In parallel with constructing the five KIAA gene-targeted mice, we isolated and characterized the full-length cDNAs of the mouse homologues of all five KIAA genes examined in the present study, because the cDNAs of the human homologues might be partial fragments of these genes. Table 1
compares the sizes of full-length cDNAs of the five human KIAA genes and the predicted amino acid sequences of their respective mouse homologues. For KIAA1409, KIAA1440, KIAA1447, and KIAA1768, the full-length proteins of the mouse homologues are extremely longer than their human counterparts, whereas for KIAA1276, the full-length mouse homologue contains 942 amino acids, many fewer amino acids than that of human KIAA1276. Human KIAA1276 contains two additional 36 aa peptides and two 74 aa peptides in the middle of the mKIAA1276 sequence; this longer form may be an alternative splicing form that exists in both human and mouse. The total lengths of mouse KIAA1409, KIAA1440, KIAA1447, and KIAA1768 were 2596 aa, 2222 aa, 2644 aa, and 1742 aa, respectively. Because we sequenced the N-terminal portions of the mouse KIAA homologues, we were able to show that they had more extensive N-terminal amino acids sequences compared with those of their human KIAA counterparts. From this comparison, we determined that mKIAA1447 and mKIAA1768 proteins contained two domains of known function. The bromo-adjacent homology (BAH) domain, known to participate in chromatin regulation and gene silencing, was identified in mKIAA1447. A motif of the guanine-nucleotide dissociation stimulator CDC25 was identified in mKIAA1768, suggesting that mKIAA1768 may be a component of the signal transduction machinery. No motifs of known function were identified in the other three KIAA proteins, KIAA1409, KIAA1440, and KIAA1276. Despite these structural findings, the overall function of all five of the KIAA proteins described here remains unknown.
Expression of KIAA mRNAs and viability of KIAA gene-targeted mice
Southern blot analysis of ES cells and tail DNA of F1 and subsequent generation offspring confirmed successful inactivation of all five KIAA genes and germ line transmission of the targeted alleles. Hybridization with both 3' and 5' probes indicated that the target alleles inserted correctly (Fig. 2
A and data not shown). Northern hybridization and RT-PCR analyses showed that intact mRNA transcribed from each of the five KIAA genes was completely disrupted at the transcriptional level (Fig. 3
A and B).
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To examine the viability of homozygotes of the five sets of KIAA gene-targeted mice, we mated heterozygotes, genotyped their pups at various stages of pre- and postnatal development, and compared the homozygotes to their heterozygote littermates (Fig. 3C
). Of the five sets of mice, two were fully viable (KIAA1276 and KIAA1768) and showed no overt phenotypes. The remaining three (KIAA1409, KIAA1440, and KIAA1447) died during embryonic development or shortly after birth. Only one KIAA1447 homozygote mouse grew to adulthood. Thus three of five sets of gene-targeted mice showed clear congenital defects during development, indicating that these three KIAA genes play an essential role in normal development.
KIAA1409–/– mice lack the ability to drink milk and die within few days
Although the large majority of KIAA1409–/– homozygote mice died at (P0) or the day after (P1) birth (Fig. 4
B), a few mice survived to P7. As shown in Fig. 3C
, homozygote and heterozygote KIAA1409 gene-targeted mice differed greatly in size and appearance. We observed milk within the stomachs of heterozygote and WT pups, but not in the stomachs of homozygote pups. Although newborn homozygote pups were active soon after birth, after half a day, the pups appeared weak and their movements decreased gradually, presumably due to their inability to consume milk [Fig. 4C and D
(movie)]. Over 90% of KIAA1409–/– pups failed to nurse. Almost no milk was found in the stomachs of dead and live homozygotes examined at P0. The remaining 10% drank very little milk in comparison to KIAA1409+/– and KIAA1409+/+ pups, the latter of which had full stomachs of milk (Fig. 4C
). Thus, unlike their heterozygote and WT littermates, KIAA1409–/–pups surviving 2–4 days after birth gained virtually no weight. KIAA1409–/– pups surviving to P6 or P7 were very thin, and their gradual weakening and ensuing death are most likely due to their inability to nurse. Placing the KIAA1409–/– pups in the care of either WT or ICR mothers did not improve this situation.
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Disruption of KIAA1440 led to embryonic lethality at the blastocyst stage
Unlike the KIAA1409–/– homozygotes, no viable KIAA1440–/– homozygotes were born (Fig. 5
A). To determine at which developmental stage these mice died, we attempted to examine their viability at E9.5, E11.5, and E13.5 of gestation, but none could be identified at these time points (Fig. 5A
). In addition, we were unable to find resorbed embryos, suggesting KIAA1440–/– embryos die very early during development, perhaps before implantation. Examination of preimplantation embryos isolated and genotyped at E3.5 revealed that KIAA1440–/– blastocysts were morphologically distinguishable from heterozygote and WT blastocysts. Thus, we concluded KIAA1440–/– embryos stop developing at the morula stage (Fig. 5B
).
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Adult KIAA1447–/– mouse had overt motor deficits
The majority of KIAA1477–/– homozygote mice died shortly after birth (Fig. 3C
and data not shown), indicating that KIAA1477 protein contributes to postnatal development of mice. Despite the high mortality of KIAA1477–/– neonates, we were able to obtain one adult KIAA1447–/– mouse. This mouse exhibited a markedly obvious phenotype. As shown in Fig. 6
A and B (movie), control heterozygote mice move about effortlessly. The KIAA1447–/– mouse, however, had great difficulty walking, often dragging its hind legs. Unlike its heterozygote littermates, the hindquarters of the KIAA1447–/– mouse were positioned abnormally, such that its loins sagged and its abdomen touched the floor.
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Quantitative assessment of motor activity in an open field test, as well as subsequent assessment of spontaneous locomotor activity, revealed significant motor/exploratory differences between KIAA1447 heterozygote and homozygote mice. Figure 6C
shows the trajectories of these mice in the open field during 5 min of observation. KIAA1447+/– mice traversed greater distances compared with that of the KIAA1447–/– mouse. Heterozygotes engaged in systematic searching behavior, covering the entire field within the allotted five minutes. On the other hand, the homozygote mouse covered only part of the field during this time. The limited scope of its search may have been due to motor dysfunction, as reflected by a gradual decrease in its searching speed, or, perhaps, to enhanced fear of open places.
Assessment of spontaneous locomotor activity with an infrared motion-sensing system revealed that the KIAA1447–/– mouse was much less active than its heterozygote littermates when placed into novel home cages (data not shown). To examine the possibility that this may be due to muscle malformation or dysfunction we examined KIAA1447 mRNA expression in the muscle of the KIAA1447–/– mouse. Northern blot analysis revealed that KIAA1447 mRNA was not expressed in the muscle of this mouse (data not shown), indicating that its decreased activity may be due to a defect in motorneurons controlling muscle, not the muscle itself. We are now attempting to raise additional KIAA1447–/– mice to adulthood by experimenting with several different kinds of breeding and housing conditions. Changing the cage size and number of mice per cage, addition of more nesting material (including cotton), and covering the cage hood all failed to affect the outcome. The majority of KIAA1447–/– pups still died between P0 and P2.
There are several possible reasons for the high mortality observed in our KIAA1447–/– mice. One explanation may have to do with the mixed genetic background of these mice: they are F2-F4 offspring of KIAA1447+/– mice with 129X1/SvJ, 129S1/Sv, and C57BL/6J backgrounds (24
25
26
27)
. Typically, the 129 substrain is used to produce ES cell lines for use in constructing gene-targeted mice, and the C57BL/6J strain is used for the extensive backcrosses required to produce isogenic knockout mice (3)
. To achieve this isogenic background, which may be necessary for KIAA1447–/– mice to grow to adulthood, we have already started to backcross our KIAA1447 gene-targeted mice with several different strains of mice with C57BL/6J and 129X1/SvJ backgrounds. Another possible explanation may be that the KIAA1447–/– mice described here possess both the mutated KIAA1447 gene and a second mutation. This second mutation may, in part, cause KIAA1447–/– pups to live or die before attaining adulthood. Further detailed analyses are ongoing.
DISCUSSION
Disruption of genes encoding large proteins causes observable phenotypic alterations because large proteins play central roles in functional protein complexes
To make progress in diagnosing and treating inherited diseases, it is necessary to identify and characterize the genes responsible for such diseases. In addition to loci mapping and disease association study, targeted mutations and gene knockouts eliciting overt phenotypic alterations provide powerful tools for studying gene function and identifying candidate genes involved in disease. A catalog of gene-targeted mice, cross-referenced according to the gene disrupted and the resulting phenotype, will greatly aid the ongoing search for genes responsible for human inherited diseases. To this end, some groups around the world have already started to collect mutant mouse phenotypes [e.g., phenotypes caused by chemical mutagens (ENU), random insertions into ES cells, and transposon mutagenesis; 28
29
30
]. These collections, however, mainly comprise phenotypes of mutant mice, and in only a few cases were the phenotype and the genes responsible for that phenotype simultaneously analyzed. Inability to preselect a particular gene and the mutated region within that gene are key weaknesses of these studies. The ideal phenotypic collection, therefore, would comprise phenotypes along with a listing of the specific genes or genetic loci causing those phenotypes. Thus, we chose specific KIAA genes, deleted specific regions within these genes to ensure the functional disruption of their protein products, constructed mice harboring these mutated genes, and then assessed the phenotypes manifested by these gene-targeted mice with the long-term aim of compiling a comprehensive database of phenotypes resulting from the targeted disruption of specific human genes.
One strength of the present study is that we were able to select specific, previously characterized genes and disrupt specific exonic regions within these genes, before expressing them in mice. There are several advantages of constructing gene-targeted mice in this controlled and orderly manner. First, one can ensure that a specific gene of choice is disrupted. Second, one can ensure that a particular region of this gene is disrupted according to its exon-intron organization. Third, because the expression profile of the targeted gene may already be known or, if unknown, can be analyzed easily, one can narrow down which tissue(s) or organ(s) to examine first. Fourth, because the chromosomal location of the gene is presumably already known, linking the aberrant mouse phenotype and its causative gene to the symptom(s) of certain congenital diseases is facilitated. Fifth, because the structure of the protein encoded by this gene is presumably already known, specific antibodies against the protein can be made, and with these antibodies, one can reliably estimate the function of the protein by determining its subcellular localization. Information about its amino acid sequence will also support functional findings based on biochemical and localization analyses, mRNA expression profile analyses, and yeast two hybrid screening or tandem tagged purification analyses. Identifying the protein-protein interactions of the protein of interest will add further to determining the function of the protein.
A sixth and final advantage of constructing gene-targeted mice is that this efficient approach avoids the pitfalls and costly consequences of constructing supposedly "mutant" mice via the phenotype-driven mutagenesis method. With gene targeting, the researcher begins with a specific "target" gene and then identifies the phenotype caused by that gene (31)
. By contrast, with phenotype-driven mutagenesis, the researcher begins with a specific "target" phenotype and then identifies the gene responsible for that phenotype (31)
. Typically, very large numbers of mice are needed for phenotype-driven mutagenesis experiments, since extensive cross-breeding between "mutant" and WT mice is required even before candidate genes can be identified. Moreover, because the entire genome is exposed to compounds that cause random mutations, one cannot avoid repeatedly obtaining mutants of previously characterized genes. Thus, large research facilities and extensive funding are required to carry out phenotype-driven mutagenesis experiments. On the other hand, even relatively small research facilities can carry out comprehensive functional genomics analyses in gene-targeted mice prepared via our approach, because excessive time and costs devoted to the caring for and the screening of mice required in phenotype-driven mutagenesis research are avoided.
At present, the difference between a gene that expresses a clear phenotype and a gene that fails to express a clear phenotype is unknown. When a gene is disrupted and subsequently expressed in a mouse, the gene-targeted mouse often appears outwardly normal. To avoid wasting time in constructing such mice, it is very important to be aware of which genes, even when disrupted, express normal-looking phenotypes. There are numerous potential reasons for the lack of discernible phenotypic alterations in gene-targeted mice. With respect to the points concerning gene compensation and hidden or minute phenotypic alterations, because large proteins possess specialized functions, compensation by other proteins is unlikely. Hidden or minute phenotypic alterations are also unlikely because the disruption of a large protein would affect the overall function of protein complexes comprised of many proteins (Fig. 7
). For these reasons, we hypothesize that the disruption of a gene encoding a large protein results in obvious phenotypic alterations in a gene-targeted mouse because large proteins play a central role in the function of protein complexes.
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Because future analyses using gene-targeted mice will likely use uncharacterized genes, the risk will increase significantly that gene-targeted mice lacking distinct phenotypic alterations will be obtained. By using our gene selection criteria, however, a researcher can lower this risk by avoiding most factors that contribute to lack of discernible phenotypes in these mice. Since our success rate is sufficiently high, it would be worthwhile to use despite the possibility of failure for the aforementioned reasons.
Concluding remarks
In summary, we used a systematic, rapid, and efficient approach to assess the function of genes that encode large proteins in transgenic knockout mice. Of the five different sets of KIAA gene-targeted mice constructed, the three harboring knockouts of the largest genes showed distinct phenotypes, indicating that KIAA1409, KIAA1440, and KIAA1447 are essential to normal development. The high success rate in our study has important implications for the future functional study of all genes in general but particularly those that encode large proteins. However, this high success rate is not unbiased, since we do not know whether disruption of any of the KIAA genes studied resulted in unmeasurable phenotypic effects, and, as of yet, no data from previous studies exist to substantiate our findings. Moreover, our approach brings a new perspective to the world of biology: a protein complex can be thought of as being organized hierarchically in terms of function. Some proteins are essential for the proper functioning of and integrity of the complex; others are relatively less important, taking on modifier roles. We propose that extremely large proteins are the key element of a protein complex because they function as the framework around which the complex is assembled. In this study, we made five gene-targeted mice, each harboring one of five KIAA genes encoding proteins 2596 aa, 2222 aa, 2644 aa, 1742 aa, or 942 aa in length. The gene-targeted mice with disruptions of the three largest proteins exhibited clear phenotypes, but mice with disruptions of the relatively smaller KIAA proteins failed to exhibit observable phenotypes. This result is in agreement with the hypothesis that we are advocating. This report represents the first comprehensive, functional assessment of proteins of unknown function derived from a cDNA sequencing project. We will continue our experiments with the goal of increasing the number of phenotypically verifiable KIAA knockout mice in line with our hypothesis that large proteins with multiple binding domains play central roles in functional protein complexes, the disruption of which results in clear phenotypic manifestations.
ACKNOWLEDGMENTS
We thank E. Suzuki, S. Minorikawa, S. Tsuda, and Y. Suzuki for excellent technical assistance. We also thank Dr. A. Pandey (Johns Hopkins Univ.) for critical reading, all our colleagues from the Kazusa cDNA Project Team for cooperation, and T. Hasegawa of the animal facility at Research Center for Allergy and Immunology (RIKEN) for taking excellent care of the mice. This study was supported by grants from the Kazusa DNA Research Institute and in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government.
Received for publication February 20, 2006. Accepted for publication March 31, 2006.
REFERENCES
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