Index References

Gene targeting is a powerful technique to analyze gene function and the pathogenesis of human genetic diseases. Embryonic stem (ES) cells derived from the 129 mouse strain have been most widely used because they have an efficient germline colonizing ability. There are, however, a number of sub-strains, such as 129/Ola, 129/J. 129/Sv and 129/ReJ, which have extensive genetic variability resulting from deliberate or accidental outcrossing. (Simpson et al., 1997; Threadgill et al., 1997). Such polymorphisms appear to be the cause of the reduced frequency of homologous recombination. Therefore, use of genomic clones derived from the ES line used for targeting experiments is desirable for complete matching of the sequences of the homology arms.

Bacterial artificial chromosome (BAC) genomic libraries are a powerful resource not only for genome analyses but also for targeting vector construction. Mouse BAC libraries derived from various strains have been constructed, including from 129 sub-strains such as 129S6/SvEvTac (Osoegawa et al., 2000) and 129S7/SvEvBrd (Adams et al., 2005). However, we can not currently access the 129/Ola BAC library via the internet. Since the 129/Ola-derived E14.1 (Kuhn et al., 1991) and E14tg2a (Hooper et al., 1987) cell lines, both of which have male genotype, are most frequently used for ES cells in gene-targeting experiments in our laboratory, we constructed a new 129/Ola BAC library.

The BAC library mixture was constructed by Advanced GenoTechs Co., Tsukuba, Japan, using E14.1 ES cells cultured without feeder cells. High molecular weight E14.1 DNA in the gel plug was partially cleaved with Mbo I, and subjected to pulse-field gel electrophoresis (PFGE) to obtain size-fractionated fragments. Three fractions of the DNA fragments, namely fraction A (around the 90-kb region), B (around the 130-kb region) and C (around the 150-kb region), were excised from the gel and re-fractionated using PFGE (Osoegawa et al., 1998). Size-selected DNA fragments were recovered from the gel and ligated to the Bam HI-digested pBACe3.6 vector (Frengen et al., 1999). The ligated mixtures were then transformed into DH10B Escherichia coli cells to generate the BAC library mixtures, which were streaked onto LB plates containing 12.5 μg/ml chloramphenicol. Single colonies were then manually placed into the individual wells of 384-well microtiter plates (Advanced GenoTechs Co.), which contained LB medium with 7.5% glycerol and chloramphenicol (12.5 μg/ml), using the same procedure we previously used to construct a medaka cosmid library (Ohtsuka et al., 2002). A total of 157,440 colonies were put into 410 plates (30,720 clones from fraction A [plates 001–080], 105,216 clones from fraction B [plates 081–354] and 21,504 clones from fraction C [plates 355–410]), which were incubated at 37°C overnight and stored at –80°C as the original plates. They were also replicated twice: one replica was stored at –80°C and used as the working plates, and the other was used to make DNA pools for PCR screening. Approximately 96% of the picked clones survived the overnight incubation.

The average insert sizes of the BAC library were estimated by PFGE analysis (21 clones) or by BAC end sequencing (95 clones). Insert sizes were 79 kb, 112 kb and 157 kb on average for fraction A-, B- and C-derived BAC clones, respectively (Fig. 1). Clones carrying no insert (7.7%, 7.7% and 28.9%, respectively) were excluded from this analysis. Considering the number of BAC clones derived from each fraction (a total of 27,223, 93,238 and 14,668 clones carrying inserts, respectively), we calculated that the average insert size of the whole BAC library consisting of 135,000 clones with insert would be 110 kb, covering 5.5 haploid genome equivalents. The probability of finding a BAC clone containing a target region was calculated to be 0.996, based on the formula of Clarke and Carbon (1976), indicating that most of the genomic region could be expected to be present in this BAC library.

View Details

Fig. 1. Distribution of insert size in BAC clones. Insert sizes of randomly selected clones, derived from fraction A, B and C, were determined by PFGE analysis or by BAC end sequencing. The average insert sizes were 79, 112 and 157 kb for fraction A, B- and C-derived BAC clones, respectively, indicating a mean insert size of 110 kb for the whole library.

The PCR screening of a high-molecular weight DNA library is very simple and it is easy to isolate target clones (Green and Olson, 1990). We extracted a total of 410 BAC DNA mixtures from each of the 384-well plates, using an alkaline-SDS method. PCR analysis using serially diluted DNA mixtures demonstrated that each mixture contained enough BAC DNA templates to perform more than 20,000 PCR reactions (data not shown). Portions of the DNA mixtures from 41 plates were mixed to generate a total of 10 DNA super-pools that were used for the first screening. Each super-pool comprised BAC DNA derived from approximately 13,500 individual clones. The PCR screening strategy for this library consisted of three steps (Fig. 2) and only 91 PCR reactions were required to determine a positive clone, which was completed within 2 days. In addition, the first PCR screening was able to identify within half a day whether or not the library contained positive BAC clones. So far, the first screening has identified a total of 8 loci, including the ROSA26 locus (Fig. 2). All markers examined were amplified in at least 4 PCR products by the first PCR screening (Table 1), which fulfilled our expectation that the probability of finding any genomic sequence was 0.996.

View Details

Fig. 2. Schematic diagram of the three steps of the PCR screening procedure for each bacterial artificial chromosome (BAC) clone. We used the primers shown in Table 1 to successfully amplify a fragment of the ROSA26 locus (Soriano, 1999). Ten DNA super-pools, each derived from approximately 13,500 independent BAC clones, were used as the PCR templates for the first PCR screening: super-pools 3, 4, 7 and 9 are positive. In the first screening step, ten PCR reactions are required. Lane 1, shown as E14.1, indicates a positive control using genomic DNA isolated from an ES cell. The second PCR screening, which requires 41 PCR reactions, was conducted for the DNA mixtures isolated from each 384-well plate. Mixtures isolated from plates 124 to 164, which were included in super-pool 4, were examined and a positive plate (plate 124 in this figure) was identified. The third screening was performed against cultured Escherichia coli mixtures using plates with vertical and horizontal grooves (Ohtsuka et al., 2002). The required BAC clone is identified by crossing the results of the vertical and horizontal lines of the 40 PCR reactions per plate (1-24 vertically and A-P horizontally). For this step, two replicas are necessary to obtain PCR templates for the vertical and horizontal grooves. Here the 124I18 BAC clone was positive. The time required for screening of the BAC clone (which also included incubation for the replica cultures) was 2 days. A total of 91 PCR reactions were required to obtain one positive BAC clone.

View Details

Table 1. Markers used for the first PCR screening

In summary, we generated a mouse 129/Ola BAC library consisting of more than 135,000 clones with 5.5-fold genome coverage. The mean insert size is approximately 110 kb. PCR screening enabled us to detect target BAC clones within 2 days. We believe that this library will be a useful resource for gene targeting studies using E14 ES cells as well as for genome analysis. Using it in combination with a recently developed recombineering technique (Liu et al., 2003; Zhang et al., 1998), we have successfully constructed several targeting vectors within 3 weeks for each vector (data not shown). Information concerning BAC clones and DNA pools developed in this study will be available from the Japanese Collection of Research Bioresources (JCRB) Gene Bank (http://genebank.nibio.go.jp/gbank/index_e.html). To obtain BAC clones containing regions of interest, PCR screening and delivery of clones preserved as agar-stab cultures will be available through JCRB as a service with charges.

We would like to thank Dr. Ei-ichi Soeda (Advanced GenoTechs Co.) for advice about the PCR screening system. Our thanks also go to Drs. Klaus Rajewsky (Harvard Medical School) and Yoichiro Iwakura (Tokyo Univ.) for providing the E14.1 ES cell lines. We are most grateful to Dr. Jun Kusuda (JCRB) for his kindness and comments on the deposit of our clones to JCRB. We thank Dr. Jennie Hui (University of Western Australia) for critical reading of manuscript. This work was supported in part by a Grant-in-Aid for Scientific Research (16651102) from the Japan Society for the Promotion of Science (JSPS).