LB and SOC medium containing 20 μg/mL chloramphenicol (Cm), and 25 μg/mL kanamycin (Kan) were used to grow BAC clones.

All bacterial cultures in this study were maintained (unless specified) at 32°C because of the temperature-inducible prophage in DY380 bacterial strain [15]. This strain was kindly provided by Dr. N.G. Copeland and contains a defective prophage to supply the expression of Exo, Bet, and Gam necessary to execute HR.

BAC DNA minipreps were purified using the Autogen-940 robot. For fingerprinting, 0.5 μg of DNA was digested with 1-2 units of EcoRI. EcoRI digests were analyzed on a 1% agarose gel in 0.5xTBE at room temperature, 4 V/cm for 8 h. The gels were stained with ethidium bromide for visualization.

pTARBAC2.1 [16] digested with EcoRI and dephosphorylated with CIAP was used as a cloning vector to construct the library and pCYPAC1 [17] as a template for kanamycin PCR amplification. We used a human BAC (RP11-622D14) containing the complete human β-globin locus. This BAC spans position chr11:5218918-5406749 hg19.

The preparation of recombination proficient orangutan genomic library followed was performed following the cloning approach developed in our laboratory [18]. DNA was isolated from a blood sample from a male Sumatran orangutan (“Segundo”) by embedding DNA in agarose. After pre-electrophoresis, high-molecular weight genomic DNA was partially digested with a combination of EcoRI restriction enzyme and EcoRI Methylase and the resultant fragments were subjected to double-size fractionation by pulsed field gel electrophoresis. DNA fragments from the appropriate size fraction were cloned into the EcoRI digested and dephosphorylated pTARBAC2.1 vector. The ligation products were then transformed into DY380 electrocompetent cells; glycerol stocks of nonarrayed library were made before library amplification on agar plates. To perform control experiments 16,000 colonies (0.7x redundant library) were picked and arrayed into 42 × 384-well microtiter dishes. Same 42 × 384-well microtiter dishes (16,000 clones) were gridded onto one 22 × 22 cm nylon high-density filter stamped in duplicate for screening by probe hybridization. To create a supply of nonarrayed library and ensure that each clone is equally represented before amplification, 10 μL aliquots were collected from 16,000 wells into one flask and snap frozen in a dry ice ethanol bath. After the titer was determined (3 × 10⁸ cfu/mL) a 1.6 × 10⁴ cfu aliquot was taken for genomic library amplification experiment.

To amplify the genomic library 16,000 clones were plated at a density of ≈3,000 cfu on 5 Petri dishes (15 mm diameter) containing Cm-agar. Plates were incubated at 32°C for 18–24 hours to assess amplification rates and representation of the primary library. To harvest the small (0,2–0,5 mm) colony growth, 7 mL of LB media was poured on the agar's surface and incubated at room temperature for 10 min on a rocking platform and 30 mL of LB containing lifted colonies were collected. Glycerol was added up to 10%, 30 × 1 mL aliquots were snap frozen and stored at −80°C with titer of 3 × 10⁸ cfu/mL or 9 × 10⁹ cfu in total.

For 10 electroporations approximately 0.6 mL (≈3 × 10⁸ cells) of an overnight culture of DY380 cells (or 3 × 10⁸ cells from the amplified library) was inoculated into 60 mL of SOB and were grown in 1 L baffled conical flasks shaking at 320 rpm until an OD₆₀₀ = 0.6 was reached. Cells were collected by centrifugation at 5500 g for 5 min, washed with ice-cold water 3 times and finally resuspended in 200 μl of ice-cold sterile water. Electroporations were performed with a mix of 2 μl of DNA and 20 μl of cells (1.5 × 10⁸ cells/μl or 3 × 10⁹ cells per electroporation) using a Gibco BRL Cell-Porator set at 315 Volts, 350 μF, 2 KΩ with resulting pulse more than 1.88 KV. After electroporation cells were resuspended in 500 μl SOC and incubated at 32°C for 1.5 hour with shaking and spread on selective agar media. The achieved efficiency of transformation for large (180-Kb) BAC molecules was 10⁷ cfu/μg of DNA.

Electrocompetent cells for HR experiments were made from temperature-induced cultures. Induction of λ-recombination of the culture for a total of 15 min at 42°C was performed following a protocol reported by Yu et al. [19]. About 1 mL (3 × 10⁸ cells) of glycerol stock from amplified library was inoculated into 60 mL of SOB media with chloramphenicol and grown at 32°C with shaking in a baffled flask for approximately 4-5 hours until an O.D.₆₀₀ = 0.6 was reached. The flask containing the culture was then placed in a water bath shaker at 42°C for 15 min (300 revs/min). Immediately after, the flask was swirled in ice-water slurry for 10 minutes to cool down. Cells were collected and washed following procedure for preparation and transformation of electrocompetent cells hereinabove.

The 1.4-Kb kanamycin cassette was PCR amplified from NotI/EcoRI linearized pCYPAC2N vector using PAGE-purified chimeric oligos. Their structure is shown in Table 3 in Supplementary Material available online at doi:10.1155/2011/560124. Each chimeric primer is 69-70 bases long and contains a portion (shown in upper case characters, 49-50 nt long) homologous to the sequence of the targeted region and a portion homologous to the kanamycin gene (shown in lower case characters, 20 nt long). The PCR products were DpnI digested and purified by washing with water using Centricon spin columns (Millipore). PCR colony screening was performed with 19–21 nt-long screening primers (Table 3). These primers yield PCR products in the range of 300–700 bp from unmodified gene, while insertion of the kanR cassette should yield a PCR product over 1.7-Kb (Figure 6). The same flanking primers were used for verification of library representation after amplification and to generate the probes for library screening by hybridization.

An aliquot from the amplified library (13.5 × 10⁸ cells) was used to prepare 900 μl of electrocompetent cells induced for recombination as described above. Nine kan^R targeting constructs were electroporated 5 times each with 0.1 μg of purified PCR product and incubated in SOB at 32°C for 1.5 hours before plating on kan LB plates.

For details of hybridization of high density filters see http://bacpac.chori.org/highdensity.htm.

In order to asses and optimize the efficiency of homologous recombination, we used RP11-622D14 BAC clone (insert size 187 Kb) which contains the complete human β globin locus. The BAC was transferred into DY380 cells by electroporation. A kan^R recombination cassette containing 50 bp terminal sequences homologous to nearby locations in the β-globin gene was amplified by PCR as previously described in [20]. The BAC clone was electrotransformed using various amounts of PCR product over a 50-fold range. The number of kanamycin-resistant colonies increased proportionally with the amount of PCR product: 250–500 clones for 10 ng; 5,000–10,000 clones for 100 ng; 20,000–25,000 clones for 500 ng of linear DNA. The 100 ng linear DNA was selected for further transformation experiments as an arbitrary compromise between recombination efficiency and total number of recombinant colonies.

To assess the selectivity of the recombination process, mixtures of the “globin” BAC with unrelated BACs from the “RPCI-11” library (http://bacpac.chori.org/hmale11.htm) were created and propagated in DY380 cells at various levels of complexity. Following the induction of HR at 42°C, electrocompetent cells for the pooled clones were prepared. The results of the transformation of these pools with the recombination cassette are shown in Table 1. The most complex mixture of BACs still resulting in 1-2 kanamycin positive clones had 10,000 unrelated BAC clones with an average 180 Kb insert size. This BAC mixture (1/10⁴) corresponds to a complexity of 1.8 Giga base pairs(Gbp), representing about 60% of the haploid human genome. This 10⁴ efficiency of homologous recombination makes our method compatible with HR screening of full size mammalian genome BAC libraries.

We took 40 random kanamycin positive clones obtained in the recombination selection experiment as a representative sample from different complexities listed in Table 1. All clones tested positive by Southern blot (data not shown) for “globin” gene(s) confirming high selectivity of homologous recombination. Subsequent PCR analysis of these clones revealed various integration sites. Figure 1(a) shows results from PCR screening of various kanamycin resistant clones using primers flanking expected β-globin integration site. Recombinant clones displayed complex recombination events as a result of a partial homology of recombination cassette with duplicated sequences within the globin locus (Figure 1(b)); All clones recovered after recombination selection originated from RP11-622D14 BAC indicating high selectivity for HR.

To test the feasibility of HR screening of BAC libraries using multiple probes, we prepared a BAC library from genomic DNA of Sumatran orangutan (Pongo abelii) in the conditional-recombination E. coli strain DY380. We then transferred 16,000 clones with an average size of 130 Kb (2 Gb total or equivalent to 0.7-fold genomic redundancy) (Figure 2) into 384-well plates for size distribution analysis and preparation of high-density filters.

To analyze genomic marker representation in the library, the arrayed clones were gridded onto a single nylon membrane. We screened the library using the traditional hybridization method with radioactive probes. Twenty-eight randomly chosen “overgo” genomic probes [21] were designed in correspondence of genomic regions highly conserved between human and mouse. Such probes were radioactively labeled and used to screen the high-density filters made from the orangutan test library. The hybridization results reveal 16 positives each from a different probe, indicating that the library is ~0.6-fold redundant. The consistency of hybridization results with the 0.7-fold estimate based on clone numbers and insert sizes argues for the absence of a major cloning bias in the DY380 strain.

Because the DY380 is a conditional temperature inducible recombination E. coli strain, it is conceivable that some leaky expression of the lambda recombination genes occurs at 32°C and that this may destabilize the BAC clones. To test for genetic instability, 8 isolates for 96 BAC clones have been fingerprinted with EcoRI. Fingerprinting data was used for an automated contig assembly in FPC to bin the clones [22]. We anticipated that for each clone, the 8 isolates fingerprinted would bind together into a contig, so we would expect 96 different contigs (1 per clone) with 8 clones per contig. For 90/96 contigs that was true, but there were a number of exceptions: for 3 clones all 8 isolates contained only a single band, 2 clones were identical, 2 clones failed to grow and in one isolate of clone#2 single band was missing (Figure 3). High-resolution fingerprinting showed that there were no significant differences between the clones in the EcoRI fragments. Therefore, we saw no evidence of major rearrangements of BAC clones during their propagation in DY380 strain in the liquid culture demonstrating genetic stability of the BACs in this strain.

The goal of library amplification is to have enough starting bacteria for preparing competent cells for transformations. For the screening with multiple HR probes (30–300), the nonarrayed library has to be amplified about 10⁴–10⁵-fold without loss of genomic representation. We amplified the library on solid agar making sure that colonies have similar growth conditions, thus maintaining the representation of the nonarrayed BAC library. By plating the library on solid agar (see Section 2 for the details), we achieved 5.6 × 10⁵ amplification rate and collected a total of 9 × 10⁹ cfu. It is noteworthy that with this amount of bacteria one could prepare up to 6 mL of competent cells—enough for ~300 electroporations with 300 HR probes.

The genomic representation of the amplified library was evaluated by PCR analysis of various titers of amplified and nonamplified library for 10 probes. Table 2 illustrates that there was no differential growth during library amplification for any of 10 probes analyzed. Hence, we achieved 5.6 × 10⁵-fold of nonarrayed BAC library amplification without a loss of genomic representation.

To screen the library by homologous integration of a kan^R recombination cassette into the genes of interest, nine target cassettes were created by PCR amplification of the kan^R gene with 70 nt chimeric oligos. The nine target sequences were a subset of the sixteen conserved regions already confirmed to be present in the library by probe hybridization. Previous reports [23] have demonstrated higher efficiencies of recombinational deletions versus insertions. To make use of this effect while minimizing the size of the deletions, both ends of targeting cassette were separated in the genome by a small distance of 50–200 bp. Hence, homologous integration of the cassette was anticipated to result in a small deletion of genomic target DNA with a concomitant insertion of the 1.3-Kb kan^R selection marker.

A stock of amplified nonarrayed library was produced by pooling together 10 μl aliquots from each well of arrayed library (16,000 clones—160 mL, ~5 × 10⁸ cfu/mL) and one of the aliquots was used to produce electrocompetent cells.

For each of the nine HR probes five 20 μl aliquots of electrocompetent cells were transformed with 100 ng of targeting cassettes (45 electroporations total) and plated on selective media. The efficiency of HR screening varied between the probes. We obtained between 1 to 10 kanamycin positive colonies for 7 cassettes. The kan^R colonies were confirmed to contain the correct insertion of kan^R gene by PCR using screening primers flanking the site of integration (Figures 4(a) and 4(b)). Because all recombineering oligos were of the same length and similar GC content, differences in HR efficiency can be attributed to specific characteristics of the targeting site such as DNA secondary structure or replication asymmetry.

The integrity of targeted BACs after recombination was verified by EcoRI fingerprinting (Figure 5). The fingerprints of the recombinant kan^R BAC clones were compared with the original BAC clones after EcoRI digestion. The EcoRI bands containing the 1.3-Kb kan^R insert have not been identified for every clone although subtle upward band shifts can be seen in some of the target products (i.e., lanes 1, 2 K, and 3 in Figure 5). Thus, there were no large rearrangements detected confirming the integrity of BAC clones after screening via HR.