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© 2002 Oxford University Press
Nucleic Acids Research, 2002, Vol. 30, No. 10 e41
A reliable and efficient method for deleting operational
sequences in PACs and BACs
Ravi Nistala and Curt D. Sigmund1,*
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
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
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.
*To whom correspondence should be addressed. Tel: +1 319 335 7604; Fax: +1 319 353 5350; Email: [email protected]
e41 Nucleic Acids Research, 2002, Vol. 30, No. 10
PAGE 2 OF 6
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.
MATERIALS AND METHODS
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 CAMR gene: primer Nhe3-F, 5′-TAGAGCTAGCATAACTTCGTATAATGTATACTATACGAAGTTATGTTGATACCGGGAAGCCCTGGGCCA-3′ and primer
Nhe3-R, 5′-GATAGCTAGCATAACTTCGTATAGTATACATTATACGAAGTTATAGGCGTAGCAACCAGGCGTTTAAGGGC-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–CAMR–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.
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
PAGE 3 OF 6
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, 0.5× 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.
Nucleic Acids Research, 2002, Vol. 30, No. 10 e41
RESULTS
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
e41 Nucleic Acids Research, 2002, Vol. 30, No. 10
PAGE 4 OF 6
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.
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
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).
using rare cutting enzymes indicated there were no other
rearrangements in PAC structure except for the insertion of the
CAM gene (data not shown).
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
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 tissuespecific, 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
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Nucleic Acids Research, 2002, Vol. 30, No. 10 e41
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.
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/counterselection 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).
e41 Nucleic Acids Research, 2002, Vol. 30, No. 10
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’ CAMR 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.
PAGE 6 OF 6
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