Nucleic Acids Research

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Nucleic Acids Research, 2002, Vol. 30, No. 10 e41
© 2002 Oxford University Press

A reliable and efficient method for deleting operational sequences in PACs and BACs

Ravi Nistala and Curt D. Sigmund^1,*

Genetics Interdisciplinary Program and ¹Departments of Internal Medicine and Physiology & Biophysics, 2191 Medical Laboratory, University of Iowa College of Medicine, Iowa City, IA 52242, USA

Received December 28, 2001; Revised and Accepted March 10, 2002.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
P1-derived artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) have become very useful as tools to study gene expression and regulation in cells and in transgenic mice. They carry large fragments of genomic DNA (100 kb) and therefore may contain all of the cis-regulatory elements required for expression of a gene. Because of this, even when inserted randomly in the genome, they can emulate the native environment of a gene resulting in a tightly regulated pattern of expression. Because these large genomic clones often contain DNA sequences which can manipulate chromatin at the local level, they become immune to position effects which affect expression of smaller transgenes, and thus their expression is proportional to copy number. Transgenic mice containing large BACs and PACs have become excellent models to examine the regulation of gene expression. Their usefulness would certainly be increased if easy and efficient methods are developed to manipulate them. We describe herein a method to make deletion mutations reliably and efficiently using a novel modification of the Chi-stimulated homologous recombination method. Specifically, we generated and employed a Lox511 ‘floxed’ CAM resistance marker that first affords selection for homologous recombination in Escherichia coli, and then can be easily deleted leaving only a single Lox511 site as the footprint.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The presence of large genomic inserts in yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) have made them the ideal choice in genomics and as tools in understanding gene structure and expression. The high level of chimerism and the difficulty of handling YACs has led many investigators to favor BACs and PACs, which are stable over generations and are easy to prepare in large quantities. Several methodologies have been developed to make precise mutations to PACs and BACs, such as single base pair substitutions, deletions and additions, while others were used to knockout specific genes (1–5). Some of these procedures are limited because they require counter-selections that are not very efficient, such as chlortetracycline and fusaric acid, while others are restricted by toxic selection/counter-selection markers (sacB/ccdB). Recently, a method using ET-cloning was described, which requires the BAC to exist in phage resistant cells; however, there are reports suggesting toxicity of the constitutively expressed gam gene (6).

We have extended the utility of a method involving the use of appropriately placed Chi-sites to enhance recombination several-fold (7,8). In this method, inclusion of the kanamycin (KAN) resistance gene in between the two homology regions facilitated selection of homologous recombinants by using KAN + chloramphenicol (CAM) double selection. A major limitation of this method is that it leaves the antibiotic resistance gene in the DNA at the site of recombination. This can be a serious drawback if one wishes to make a deletion that leaves a minimal footprint (i.e. deletion of a putative regulatory element in the 5'-flanking region of a gene). We have devised a strategy to delete the antibiotic marker leaving only a Lox511 site as the footprint. In developing this strategy we recognized that PAC (pAd10sacBII) and BAC (pBeloBAC) vectors have a LoxP site in the vector backbone and thus recombination with a LoxP site within the gene of interest (i.e. floxed antibiotic resistance gene) would cause large deletions of vector and insert DNA, which would be useless. Therefore, the strategy is founded on the observation that recombination between heterotropic LoxP sites (LoxP versus Lox511) is extremely low, whereas recombination between homotropic Lox511 sites occurs very efficiently in the presence of Cre-recombinase (9,10). By placing the variant Lox511 sites on either side of the selection marker, we could specifically delete the marker by simply passaging the modified PAC or BAC clone through an Escherichia coli strain expressing Cre-recombinase constitutively. There are several advantages of this method including the availability of a wide range of cloning sites, 100% efficiency of Cre-mediated deletion and the suitability for both PACs and BACs.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Generation of the targeting construct
To construct a recombination cassette in pBluescript plasmid, roughly equally sized fragments that were homologous to DNA upstream (A) and downstream (B) of the putative renin gene enhancer were cloned by PCR. A 30 bp multiple cloning site (consisting of EcoRI–PstI–SmaI–BamHI–SpeI–XbaI) was placed between homology A and homology B. An 1.1 kb recombination cassette was ligated into the SalI site of pRM4-N (containing appropriately placed Chi-sites) to generate the plasmid pPS (Fig. 1A) (7). In addition, we also constructed a Lox511–CAM–Lox511 recombination cassette in pCR2.1 to generate the plasmid pCAMLOX (Fig. 1B). This vector provides the Lox511 ‘floxed’ CAM resistance gene. The following primers (Lox511 sites underlined) were used to amplify the 860 bp CAM^R gene: primer Nhe3-F, 5'-TAGAGCTAGCATAACTTCGTATAATGTATACTATACGAAGT-TATGTTGATACCGGGAAGCCCTGGGCCA-3' and primer Nhe3-R, 5'-GATAGCTAGCATAACTTCGTATAGTATACATTATACGAAGTTATAGGCGTAGCAACCAGGCGTTT-AAGGGC-3'. The Lox511 sequence was chosen based on the sequence of Hoess et al. (9). A mutation in the Lox511 site, described by Bethke and Sauer (10), which changes the terminal T to G and was recombinationally neutral was not used. The PCR conditions were: initial denaturation cycle at 94°C for 2 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 30 s, and extension at 68°C for 90 s. Next, a 935 bp NheI–Lox511–CAM^R–Lox511–NheI fragment was cut out and ligated into the SpeI site between homology A and homology B, to give pCLPS, the final targeting construct (Fig. 1C). All restriction sites were engineered to be at the ends of the appropriate PCR primers.

View larger version (19K): [in this window] [in a new window]   Figure 1. Schematic maps of the targeting constructs. Maps of pPS (A), pCAMLOX (B) and pCLPS (C) are shown. pPS has three appropriately placed Chi-sites on either side of each homology. pCAMLOX contains a Lox511–CAM–Lox511 cassette, which can be cut out either using NheI or any of the enzymes in the vector pCR2.1. pCLPS linearized at NotI is 4.6 kb in size.

 
DNA preparation and transformation
PAC160 was electroporated into MC1061 cells made electrocompetent with 10% glycerol to establish a stable line of MC1061 recA⁺ cells containing PAC160. Clones were selected on Luria–Bertani (LB) agar plates supplemented with 25 µg/ml KAN. Clones were checked to ensure they matched the structure of the original PAC with both six-base and rare-cutting restriction enzymes. Miniprep DNA for PAC160 was prepared using 10 ml overnight bacterial cultures in LB broth supplemented with 25 µg/ml KAN (Qiagen Tip-20 kit). Large scale preps were either made using KB100 Magnum kits (Genome Systems, St Louis, MO) or Qiagen Mega kit (Qiagen Inc., Germany). The targeting construct (pCLPS) DNA was prepared using standard alkaline lysis protocols. MC1061 cells were made electrocompetent as previously described (11). Briefly, 250 ml bacterial cultures were grown to an OD₆₀₀ of 0.7 and centrifuged at 5000 r.p.m. at 4°C in a Sorvall 5LA1500 Rotor. The supernatant was discarded and the cells were resuspended in 10% ice cold glycerol. This procedure was repeated twice and the cells were frozen on dry ice in aliquots of 500 µl. MC1061 cells containing PAC160 were made transformation competent (for the targeting vector) using the CCMB method (12). Briefly, a 50 ml culture was grown to an OD₆₀₀ of 0.3, the cells were centrifuged and resuspended in CCMB medium [80 mM CaCl₂, 20 mM MnCl₂, 10 mM MgCl₂, 10 mM potassium acetate, 10% (v/v) redistilled glycerol].

Transformation and selection of homologous recombinants
Transformation of PAC160-containing MC1061 cells was performed using a variation of the protocol described previously (7). CLPS DNA linearized with NotI was run overnight at 45 V on a 0.8% agarose gel and the 4.6 kb band was extracted using a commercially available gel extraction kit (Bio-Rad Laboratories, CA). Before transformation, 200 ng of DNA was heated to 65°C for 10 min and then cooled on ice (8). This DNA was suspended in 50 µl of MCT buffer and added to 120 µl of CCMB competent MC1061 cells. The remaining steps were as previously described (7). LB agar plates supplemented with 25 µg/ml KAN and 12.5 µg/ml CAM were used to select for homologous recombinants. The clones growing on LB + KAN + CAM were then screened on LB + ampicillin (AMP).

Analysis of recombinants
PCRs were performed using standard 1.5 mM Mg concentrations and Taq polymerase (Roche Diagnostics Corporation). PCR primers were: primer a, 5'-AGAGAAAGGGTGGGTGGTCA-3'; primer b, 5'-GCAGGGCTGGTGGGAACA-3'; primer c, 5'-CCGCTCTAGAGCCAATCTTTTCTAATGAT-3'; and primer d, 5'-GGTGGAATTCAGGGGATAGATGTGGGAGTG-3'. PCR with primer a and primer b was performed in a 100 µl reaction for 35 cycles (94°C, 1 min; 56°C, 1 min; 72°C, 1 min) preceded by one denaturation cycle for 2 min. PCR with primer pair a and c, or primer pair b and d was done at the same time using 100 µl reactions for 35 cycles (94°C, 1 min; 56°C, 1 min; 72°C, 3 min) preceded by one denaturation cycle for 2 min. Southern blot was performed on PAC DNA digested with SmaI and homology A was used as a ³²P-labeled probe. Standard protocols were used for the hybridization and washing of the membrane. Field inversion gel electrophoresis (FIGE) was done using Program 3 on a BioRad FIGE mapper. Briefly, a 1% pulse-field certified agarose gel was run under the following conditions: 16 h at room temperature, 0.5x TBE buffer, switch time ramp 0.1–2.0 s, forward voltage 180 V and reverse voltage 120 V. The gel was stained with ethidium bromide for 20 min and destained for 30 min.

Deletion of CAM^R (CAM) gene
One clone (clone 8), also referred to as P–E+C (PAC minus enhancer + CAM), was selected for electroporation into BS591 cells (13). BS591 cells were made electrocompetent using the same protocol as for MC1061 cells. Fifty microliters of BS591 cells were electroporated with 0.5 µg of P–E+C and the clones were selected on LB + KAN. Clones were screened for CAM sensitivity by replica tooth picking onto LB + KAN and LB + CAM plates. PCR was performed to verify deletion of the CAM marker using bacteria lysed in 10 µl of TE and the reaction mixture brought to 100 µl. Southern blot analysis and FIGE were done as before. Restriction analysis was done using 1 µg of PAC DNA and run overnight at 25 V on a 1% agarose gel. Sequencing was done using the dye termination method in the DNA core facility at the University of Iowa.

The final modified PAC was transferred into DH10B cells (recA^–, Cre^–) for permanent storage of the modified PAC.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We wanted to use homologous recombination in bacteria as a rapid, reliable tool to make a precise deletion of the enhancer located upstream of the human renin gene located on a PAC (14). For this purpose, we obtained a PAC from Genome Systems with a 160 kb insert containing the human renin gene, 75 kb of DNA on the 5' flank, and 70 kb in the 3' flank. The enhancer is 241 bp in size and is located 12–13 kb upstream of the renin transcription start site (15). The enhancer has been shown to influence reporter gene transcription in As4.1 cells, and is implicated to participate in the tight regulation of renin gene expression and regulation observed in transgenic mice containing PAC160 (14,16). However, the in vivo role of the enhancer has not been directly tested. Hence, we chose to use a novel strategy to delete the enhancer upstream of the renin gene and eventually develop transgenic mouse models in order to study the importance of this cis-acting regulatory element in renin gene regulation.

To build a targeting construct for homologous recombination, we used a vector that contains three appropriately placed Chi-sites on either side of the cloning cassette. Chi-sites stimulate a 50-fold higher recombination frequency in bacteria (8). First, a recombination cassette, containing homologous DNA located upstream (A) and downstream (B) of the enhancer was cloned into this vector, and a 30 bp multiple cloning site was included between the two homologies (Fig. 1A). We also generated a recombination cassette containing the floxed CAM gene (Lox511–CAM–Lox511, pCAMLOX) which first affords selection for homologous recombinants, and can then be deleted using Cre-recombinase (Fig. 1B). The floxed CAM gene was ligated between homology A and homology B, to generate pCLPS, the final targeting construct (Fig. 1C).

pCLPS was cut with NotI thus eliminating the 700 bp pUC ori and transformed into MC1061 cells containing PAC160. Clones were selected on LB agar plates containing both KAN and CAM (Fig. 2A). Recent reports suggest that the recA gene induces instability in PACs/BACs, but our observations suggest that no modification occurs once an intact PAC/BAC is stably in place within the cell. Nine clones growing on KAN and CAM were screened for AMP sensitivity, and one clone (clone 8) was found to be KAN and CAM resistant but AMP sensitive. Clone 8, the potential homologous recombinant and a clone (clone 6) that was resistant to all three antibiotics were selected for further analysis. PCR of clone 8 with primer set a + b confirmed the increase in size consistent with the insertion of a 965 bp DNA fragment containing the floxed CAM gene in place of the 241 bp enhancer (data not shown). PCR of clone 8 with primer sets a + c and b + d also showed the appropriate size increase confirming both the presence of the appropriate insertion and the maintenance of the junctions between the homologies and contiguous DNA upstream and downstream of the homologies (Fig. 2B). On the contrary, PCR of clone 6 gave the same sized bands as the original PAC demonstrating the absence of homologous recombination and suggesting either the maintenance of two separate plasmids or a random insertion of the recombination vector into the PAC. No bands were observed using pCLPS because primers c and d were in DNA outside the homologous regions. Southern blot analysis using common restriction enzymes and coupled with FIGE using rare cutting enzymes indicated there were no other rearrangements in PAC structure except for the insertion of the CAM gene (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 2. Selection for homologous recombination. (A) Schematic for obtaining a homologous recombinant. In brief, the linearized targeting vector CLPS was transformed into MC1061 recA+ PAC160 cells and clones were selected on KAN + CAM and screened for sensitivity to AMP. (B) Verification of homologous recombination by PCR using primer sets b + d and a + c. The location of the primers is shown in (A).

 
Once the correctly targeted clone was identified (clone 8, now P–E+C), we proceeded to delete the CAM gene from the modified PAC. BS591 cells are DH5 cells that express Cre-recombinase from a lysogenic phage integrated into the bacterial chromosome (13). Since Cre-recombinase is expressed from the chromosome we avoided the problems of Cre-plasmid contamination in our PAC DNA preps. Electrocompetent BS591 cells were electroporated with P–E+C and colonies were selected on LB containing KAN (Fig. 3A). Forty-nine of 49 colonies grown on a master plate containing KAN were sensitive to CAM demonstrating that Cre-recombinase is essentially 100% efficient in removing the antibiotic resistance marker.

View larger version (40K): [in this window] [in a new window]   Figure 3. Deletion of the floxed CAM gene. (A) Excision of CAM marker using Cre-recombinase. P–E+C was electroporated into BS591 cells that constitutively express Cre-recombinase. Cre-mediated recombination resulted in excision of the CAM marker leaving a single Lox511 site. The clones were selected on KAN and screened for sensitivity to CAM. (B) Representative PCR on 10 clones. P, PAC160. (C) Southern blot confirmation of the deletion using the indicated restriction enzymes and homology A as a probe. P, PAC160; P–E, final modified PAC clone lacking the enhancer. (D) Fingerprint analysis of the modified PAC. PAC160 and P–E were digested with the indicated restriction enzymes and fragments were resolved on an agarose gel.

 
PCR was performed on 10 clones using primer set a + b (Fig. 3B). Nine of 10 clones had a PCR product of 540 bp compared with 740 bp for PAC160 and 1.4 kb for P–E+C, fully consistent with the deletion of the 241 bp enhancer and addition of a single Lox511 site. One of the clones, now called P–E (PAC160 minus enhancer), was selected for further analysis. Southern blot analysis using homology A as a probe shows that the HindIII (flanking the enhancer) fragment size was smaller (3.7 to 3.5 kb), a new SmaI site was introduced (4.3 to 1.9 kb), and an MscI site was removed (1.7 to 2.6 kb) as a result of deletion of the enhancer (Fig. 3C). In order to determine if there were any overall structural abnormalities in this twice modified PAC, we performed a ‘fingerprint’ of PAC160 and P–E with six frequently cutting restriction enzymes. When digested with SmaI, the banding pattern for PAC160 and P–E was identical except for the anticipated shift of one band from 4.33 to 1.9 kb due to the addition of the SmaI site between homology A and homology B (Fig. 3D). The pattern of bands was identical using the other enzymes because the anticipated shifts migrate as doublets and triplets. These data indicate there are no obvious short range modifications of the PAC as a result of the manipulations. FIGE was used for long-range mapping to compare the restriction pattern for PAC160 and P–E. There were no gross rearrangements detected after ethidium bromide staining, and a renin cDNA probe hybridized to the same 45 kb NotI–SalI fragment (data not shown). Finally, direct sequencing of PAC160 and P–E was done using primer a. Comparison of the two sequences shows that while PAC160 has an intact enhancer, P–E lacks the enhancer but contains a Lox511 site.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
With the sequencing of the human genome essentially complete, a large amount of sequence information exists in the public databases regarding genes and their nearby regulatory elements. Prediction programs for transcription factor binding sites, e.g. TF search can identify short sequences of DNA that are of potential importance in gene regulation. As we showed for PAC160, large genomic segments encoded in PACs/BACs can potentially include all the cis-regulatory elements needed for the transcription of a gene(s), making PACs/BACs useful tools for gene analysis. However, there is a need for better methods to reliably and efficiently modify the important operational sequences in order to study their role in vivo. There are numerous examples where important regulatory elements identified by ‘promoter bashing’ in cell lines has provided information that could not be replicated in vivo. We reported that deletion of regulatory sequences thought critically important for angiotensinogen expression in HepG2 cells from a properly regulated genomic clone had no effect on tissue-specific, cell-specific or hormonal regulation of the gene in transgenic mice (17). Expression of human renin from PAC160 in transgenic mice leads to a tightly regulated pattern of expression which is proportional to copy number (14). Deletion of potential operational sequences in a PAC (or BAC) clone followed by examination of the expression of renin in transgenic mice should provide a novel opportunity to examine the physiological significance of these sequences in vivo.

Several methodologies exist to modify PACs and BACs. Most of these methods involve the use of selection/counter-selection strategies that are very toxic and not always efficient. The method described herein achieves homologous recombination in the first step with a high percentage of correctly targeted gene modifications obtained per transformation. Indeed, one out of nine clones were positive from a single transformation. Efficiencies of 15–20% have been reported with this method (7,8). When designing the targeting construct, we used homologies that were only 500 bp in size. Therefore, it is possible that using larger segments of homology could increase the frequency of homologous recombinants. Nefedov et al. (5) had to screen over 450 clones to obtain four positives indicating a much lower efficiency of recombination. We have also tried unsuccessfully to use the protocol developed by Yang et al. (1) which involves first generating a co-integrant between the PAC and the targeting vector followed by a selection scheme for resolution. Although we were able to obtain co-integrant clones with a reasonable efficiency, we were unable to resolve them using the chlortetracycline/fusaric acid selection protocol. Recently, a method based on ET-recombination has been reported by Zhang et al. (6). Although the PCR-based construction of a targeting construct has advantages, there are reports about the toxicity of a constitutively expressed gam gene, which lowers the efficiency of selection for a homologous recombinant (5).

For the second step, Cre-mediated excision of the CAM marker was essentially 100% efficient and very simple to carry out. With a single passage through BS591 cells, we deleted the Lox511 ‘floxed’ CAM^R marker, leaving only the Lox511 site as the footprint of the deletion process. For studies of gene regulation in response to deletion of regulatory elements, leaving the smallest possible footprint would be the most attractive as it would minimize the chances of artificially adding new transcriptional instructions. Although we cannot formally rule out the possibility that the Lox511 site will affect transcriptional regulation of the renin gene, no such effects have been reported in experiments using the Cre-LoxP system. Moreover, TFSEARCH of the Lox511 site reveals a target for the potential binding of a chicken homeobox gene (CdxA), whose recognition site was determined by selection of random oligonucleotides with GST-CdxA protein (18).

The method employed here is based on the high frequency of recombination between identical LoxP (or Lox511) sites, and the low frequency of recombination between heterotropic Lox sites. However, recent studies question the reliability of differentiating between LoxP and Lox511 sites. One of these studies investigated Cre-recombinase activity in vitro, and both reports used Cre fusion proteins which may alter the specificity of the enzyme (19,20). Of the 10 clones screened after passing the modified PAC clone through Cre-expressing bacteria, one did not amplify with primers present in the upstream and downstream homology. Analysis of that clone suggests it contained a large deletion of PAC DNA. Therefore, it remains possible this may have resulted from heterotropic recombination between one of the Lox511 sites and the LoxP site in the vector. Nevertheless, the structure of the other nine clones is consistent with a single recombination event between the Lox511 sites, suggesting the frequency of ‘illegitimate’ recombination is low.

In conclusion, as detailed information on genes and their regulatory elements increases, methodologies to analyze gene function will present a continuing challenge to investigators. Although the method described herein is intended to delete an enhancer fragment from a PAC clone, the methodology should work for other types of genetic modifications.

	ACKNOWLEDGEMENTS

 
We wish to thank Dr S. Lin and Dr Henry Paulson for the pRM4-N vector, Dr Nathaniel Heintz for providing us with the pSV.RecA vector, and Dr Brian Sauer for BS591 cells and other Cre-recombinase and LoxP containing vectors. We thank Dr Henry Keen for proofing the manuscript. We also thank Dr J. Jessen and Dr Yaohui Chai for useful discussions and suggestions.

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +1 319 335 7604; Fax: +1 319 353 5350; Email: curt-sigmund{at}uiowa.edu

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