Rapid Screening of a Novel Arrayed Medaka (Oryzias latipes) Cosmid Library

1Department of Genetic Information, Division of Molecular Life Science, School of Medicine, Tokai University, Kanagawa 259-1193, Japan; 2Department of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8601, Japan

Summary

Medaka (Oryzias latipes) has a lot of advantages for genetic and developmental studies. With recent advances in the genome analyses of other species, rapid accumulation of resources for medaka genomics is expected. In this study, we generated an arrayed medaka cosmid library from the HNI inbred strain, carrying a 40 kb insert on average. The library consists of approximately 120,000 clones with a six-fold genomic coverage. Cosmid clones can be screened within two days using standard PCR method. Considering the advantage of the cosmid insert size and compact genome size of the medaka, this library provides a powerful tool for future genome analyses.

Introduction

Medaka (Oryzias latipes) is a small freshwater fish. It has advantages for genetic and developmental studies similar to zebrafish such as small body size (3 cm in length), external fertilization and development, transparency of eggs, short generation time (2 - 3 months), facility of crossing and breeding, and established methods of creating transgenics (Ozato et al., 1986; Stuart et al., 1988) and chimeras (Wakamatsu et al., 1993; Lin et al., 1992). Furthermore, medaka has the advantages that several inbred strains such as the HNI and Hd-rR strains are available (Hyodo-Taguchi and Sakaizumi, 1993), and the genome size is small (800 Mb: one fourth that of human [3000 Mb] and one half that of zebrafish [1700 Mb]) (Uwa and Iwata, 1981). Recently, methods of nuclear transplant has been established (Wakamatsu et al., 2001). In a mutagenesis study of medaka, mutated phenotypes that could not be found in large-scale mutagenesis of zebrafish were obtained (Ishikawa, 2000). From these advantages, medaka has become increasingly popular as an experimental model.

Recently, the development of resources for medaka genomics has been progressing rapidly. Genetic linkage maps of medaka have been constructed and locations of some mutations and known genes have been identified (Ohtsuka et al., 1999; Naruse et al., 2000). The positional cloning approach and positional candidate gene approach are possible by using information on chromosome location. For these approaches, a genomic library is indispensable. Presently, there are many cloning vectors available for constructing a genome library. The BAC vector (Shizuya et al., 1992) is one of the vectors frequently used for genome analyses. A medaka BAC library recently constructed from the Hd-rR inbred strain has a 210 kb insert on average (Matsuda et al., 2001).

Cosmid library may contain about 40 kb of genomic fragment (Hohn and Collins, 1980). Although this insert size is smaller than that of BAC or PAC clones, a cosmid clone is the best choice for some purposes. Handling of a cosmid clone is easier, and the number of restriction fragments of a cosmid clone is less numerous than that of a BAC or PAC clone, making analyses or the modifications easier. Therefore, a cosmid clone is useful for detailed analyses of a restricted region. Here, we report the construction of an arrayed medaka cosmid library with a six-fold genomic coverage using the HNI inbred strain. Cosmid clones arrayed into 384-well plates, can be screened by the polymerase chain reaction method within two days. This library should be a useful tool for genomic analyses.

Results and Discussion

In the present study, the SuperCos1 cosmid vector was used for library construction. The partially Sau3AI digested and size-selected genomic DNA was ligated to vector arms. After the packaging procedure, ligated DNA was transfected into XL1BlueMR host cells with an efficiency of 9.1 ~ 106 cfu/ƒĘg insert DNA. To estimate the average insert size of this library, 61 randomly isolated clones were examined. Analysis by Eco RI and NotI digestion of these clones revealed that the average insert size was 40.2 ( 3.9 kb (Figure 3). Clones without any inserts were not detected.

A total of more than 1.2 ~ 105 clones were picked into the 384-well plates. Small colonies were picked as much as possible, to prevent bias. About 99 % of the picked clones were successfully cultured after overnight incubation. Based on average insert size and medaka genome size (800 Mb) (Uwa and Iwata, 1981), this library is believed to have a six-fold coverage of the medaka haploid genome. The probability of finding any given medaka sequence was calculated to be 0.998 using the formula ( Clarke and Carbon, 1976). Therefore, most of medaka genomic sequence is expected to be present in this library.

The PCR screening strategy for this library consists of three steps (see "Materials and methods"; Figure 1, Figure 2). Using this screening system, clones can be obtained within two days (Figure 2). Moreover, by conducting the first PCR screening, we can determine whether or not this library contains positive cosmid clones within half a day. So far, the first screenings were conducted with a total of 26 markers. All markers examined were amplified in at least one PCR product by the first PCR screening (data not shown). This result fulfilled the expectation that the probability of finding any medaka sequence is 0.998, as mentioned above. Regarding 14 of 26 markers, positive cosmid clones were identified by completing the PCR screening and all clones we examined were independent.

As described above, inserts in our clones were sufficient in size and clones lacking inserts were not detected. So far, isolated cosmid clones have been used for various analyses such as fluorescent in situ hybridization (FISH), genomic southern hybridization, cosmid walking and sequencing (described elsewhere). No confusing data, such as the presence of chimeric clones, have been detected in these analyses. We can assume from these results that the quality of this library is suitable for use in various kinds of genomic analyses.

Populations of Japanese medaka consist of two genetically distinct groups, a northern population such as HNI strain and a southern population such as Hd-rR strain (Sakaizumi et al., 1983). The degree of polymorphisms between these two populations is very high (approximately 1 polymorphism / 31 bp in the noncoding regions: Ohtsuka et al., 1999). In this study, DNA from the northern population (HNI inbred strain) was used as cosmid inserts for three reasons. First, the genetic map we constructed was based on HNI strain-specific polymorphisms, and some markers were amplified only from the northern population by the PCR method (Ohtsuka et al., 1999). Second, the BAC library was constructed using the genomic DNA derived from the southern population (Hd-rR inbred strain) (Matsuda et al., 2001). Therefore, genomic clones derived from the northern population will be useful for evolutional genetic analysis. Third, polymorphisms specific for the northern population can be used as selection markers if a cosmid clone is injected into individuals of the southern population. Most medaka mutants were isolated from the southern population, therefore transgenic rescue experiments can be performed with cosmid clones derived from wildtype northern population (HNI strain).

The genome size of the medaka (800 Mb) is approximately one-fourth that of human (3000 Mb). Because this compact genome is thought to reflect smaller intro- and inter-genic sequences, a 40 kb (cosmid size; modification of the sequences is easily done using restriction enzymes) fragment of the medaka genome would correspond to 150 kb (BAC or PAC size) fragment of the human genome. From recent progress in the human genome project, the estimated number of genes in human is 30,000 to 40,000 (International Human Genome Sequencing Consortium, 2001; Venter, J. C, 2001). If the number of genes in medaka is the same as in human, one or two genes might exist in a 40 kb fragment of the medaka genome, and one cosmid clone of medaka is expected to contain a complete gene including its regulately element. Therefore, by using cosmid clones, we can possibly analyze specific genes free from the influence of other genes residing flanking regions. From these facts, the cosmid library constructed in this study should be a powerful tool not only for the study of the medaka genome but also for the broad analyses of gene function and comparative genomics.

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