MINIREVIEW SERIES FOR THE 50TH
VOLUMEThe development and applications of the bacterial arti®cial
chromosome cloning system
Hiroaki Shizuya and Hosein Kouros-Mehr
Beckman Institute, Division of Biology, California Institute of Technology, Pasadena, CA, USA
Abstract. The development of the Bacterial Arti®cial Chromosome (BAC) system was driven in partby the Human Genome Project as a means to construct genomic DNA libraries and physical maps forgenomic sequencing. The BAC system is based on the well-characterized Escherichia coli F-factor,
a low copy plasmid that exists in a supercoiled circular form in host cells. The structural features of theF-factor allow stable maintenance of individual human DNA clones as well as easy manipulation of thecloned DNA. BACs are currently used in a wide array of applications from genome sequencing to genediscovery. This paper describes the key elements in the development of the BAC system and its current
notable applications. (Keio J Med 50 (1): 26±30, March 2001)
Key words: BAC, YAC, pBeloBAC, pIndigoBAC

The Development of the BAC Cloning System

The Human Genome Project will undoubtedly havea profound effect on our understanding of the biologicalsciences and medicine. The knowledge generated bythese efforts will change the practice of medicine interms of the diagnosis, treatment, and prevention of
disease. By the time this manuscript is published, mostof the human genome and possibly the genomes ofother organisms such as the mouse will have beensequenced. Genomic sequencing is only the beginningof an exciting and fruitful advancement in medical genetics. The post-sequencing era of functional genomics,
which will require the annotation, deciphering, and interpretation of the genomic sequence, will be more dif®cult to complete and may take a considerably longer
period of time. The annotation will require intimate
collaboration between scientists working in biologicalsciences and in information technology. Physicians will
also take more active roles in advancing medical
sciences by utilizing the information generated byfunctional genomics studies. We feel honored to havebeen a part of these endeavors from the beginning
through the Bacterial Arti®cial Chromosome (BAC)
system that we developed. The BAC system is now
widely used by many scientists in sequencing efforts aswell as in studies of genomics and functional genomics.

As the Human Genome Project was underway inthe early 1990s, there came the need to create highresolution physical maps of each human chromosome,which would permit the isolation of short DNA fragments for direct sequencing and other manipulations. In
response to this need, the Yeast Arti®cial Chromosome(YAC) system was developed.1 Although YACs can
carry DNA as large as one mb, subsequent studies
indicated that the YAC system presented several dif®culties in the creation of a human genome map.2±4 Additionally, yeast cells were not as familiar to molecularbiologists as other organisms such as Escherichia coli.
To circumvent these dif®culties, we developed a bacterial cloning system based on the well-characterized
E. coli F-factor, a low-copy plasmid that exists in asupercoiled circular form.5 We had three major goals inmind when we developed the BAC cloning system.
First, we wanted to have a stable bacterial cloning system that was easy to manipulate for mapping andgenome analysis. The long linear structure of YACs
made it dif®cult to recover the cloned DNA in pureform, as YACs were more susceptible to shearing. Our
second goal was to have a large insert that could serve

Reprint requests to: Dr. Hiroaki Shizuya, Beckman Institute, Division of Biology, 139-74, California Institute of Technology, Pasadena,
CA 91125, USA, e-mail: shizuya@its.caltech.edu
26

Keio J Med 2001; 50 (1): 26±30

as a substrate for DNA sequencing. Other bacterial
cloning systems such as cosmid and P1 vectors had beendeveloped but these had maximum cloning capacities of
45 kb and 100 kb, respectively.6 The third goal was toprovide a tool that would later be used for functional
genomic studies once the human genome and othergenomes had been sequenced.
BAC vectors
We based the BAC system on the Escherichia coli
F-factor because of the wealth of information in its
genetics and molecular biology. The F-factor had long
ago been used as a tool for studying gene regulationand mutation analysis in a variety of E. coli operons.
Replication of the F-factor is strictly controlled by theregulatory functions of E. coli, and as a result the Ffactor is maintained in low copy number (i.e., one or
two copies per cell). This allows stable maintenance oflarge DNA inserts and reduces the potential for recombination between DNA fragments carried by thevector. The latter property is re¯ected in the fact thatgenomic inserts in high-copy number cloning systems
such as cosmids often undergo extensive and unwanted
rearrangements. In addition to stable maintenance, thestructural stability of F-factors allows complex genomic
DNA inserts to be maintained with a great degree ofstructural stability in the E. coli host.
BACs have several additional advantages over
YACs. It was observed that a large percentage of
YACs carried chimeric inserts, making mapping efforts
confusing and dif®cult. BACs, in contrast, are virtually
free from chimerism. Another problem with YACs isthat multiple YAC chromosomes may coexist in a single yeast cell. In the BAC system, the F-factor-encoded
parA and parB genes are involved in exclusion of multiple F-factors, and as a result multiple BACs cannot
coexist in a single cell. We have thus far analyzed morethan 10,000 BAC clones and have not yet found cells
carrying multiple BACs.
BAC vector cloning site
The BAC vector incorporates several sites andmarkers. The cloning segment includes (1) two bacteriophage markers, lambda cosN and P1 loxP; (2) three
restriction enzyme sites (EcoRI, HindIII and BamHI)
for cloning; and (3) a GC-rich NotI restriction enzymesite for potential excision of the inserts. The cosN site
provides a ®xed position for cleavage by bacteriophage
lambda enzyme terminase, which allows the convenient
generation of a linear form of the BAC DNA. The cosNsite is also used to package approximately 50 kb DNA
into the bacteriophage lambda head as a particle. The
method, known as Fosmid for F-based cosmid system, is27

extremely ef®cient and thus very useful when DNA isprecious or available in limited amounts. The P1 loxPsite allows the retro®tting of additional components to
the BAC vector at a later stage; e.g., the addition of theG418 resistant marker for selection in eukaryotic cells.
The loxP site can also be used to linearize BACs
through the P1 phage protein Cre, which catalyzes
strand exchange between two DNA strands at the loxPsites.
The BAC vector pBeloBAC11 contains an additional component in the cloning site ± b-galactosidase(lacZ), which allows a complementation. This allows
clones with inserts to be readily identi®ed as an X-gal
color change. A more recent BAC vector known as
pIndigoBAC displays a much faster and deeper X-gal
color change as a result of a point mutation in the 3 0
end of the lacZ gene. This frameshift causes prematuretermination of the b-galactosidase polypeptide. All
BAC vectors, including pBeloBAC11 and pIndigoBAC, contain cloning sites that are ¯anked by T7 andSP6 promoters, which can be utilized for DNA
sequencing of the insert at the vector-insert junction(BAC end sequencing). Additionally, the BAC vectors
carry a chloramphenicol resistance gene in order toscreen for host cells that carry BACs.
BAC cloning
Two important enzymes were key in the creation of
the BAC system ± agarase and temperature-sensitive
alkaline phosphatase. Although we knew how to separate large DNA through Pulsed-Field Gel Electrophoresis, we did not know how to purify DNA from gel
matrices without damaging the DNA. After trying several procedures, we found that the enzyme agarase
could be used to recover the DNA by breaking down
the agarose matrices. The main problem we faced isthat there was no pure agarase that could be usedwithout damaging DNA larger than 100 kb; fortunately,an enzyme supply company aided us by providing
DNAse-free agarase. The second enzyme, temperaturesensitive alkaline phosphatase, was greatly needed toprepare BAC vector DNA. Since the BAC vector is asingle-copy plasmid, it was extremely laborious to obtain suf®cient amounts for use in cloning, especiallysince the DNA must ®rst be dephosphorylated. At thetime, the available enzyme for dephosphorylation was
E. coli alkaline phosphatase, which is extremely dif®cult
to remove from the system subsequent to the reaction
and can ultimately result in a considerable loss of DNA.
We needed a phosphatase that could be destroyed byraising the temperature to 65 C. Fortunately, we had
puri®ed such an enzyme about 10 years ago from abacterial species isolated in Antarctica, and in collaboration with the same company we obtained the28

Shizuya H and Kouros-Mehr H: BAC system development and applications

temperature-sensitive alkaline phosphatase that weneeded.
An additional obstacle was to choose the correct
bacterial strain for cloning. Although most E. coli
strains can be transfected with DNA of a maximum sizeof 100 kb, we found that certain strains of E. coli suchas DH10B or DH5 can take up DNA larger than 100
kb. There may be a genetic disposition or speci®c genetic markers in the DH10B or DH5 strains that may
be responsible for this differential uptake.
Construction of BAC libraries
Having constructed the BAC cloning system, we
subsequently made four human BAC libraries fromthree individuals.7 Each library contains BAC clones ofmultiple coverage and an average insert size of 125±175
kb. A fraction of Library A contained the original BAC
vector pBAC108L, which lacked the lacZ gene for Xgal
color selection. As a result, we needed to perform colony hybridization in order to identify clones carrying
human DNA inserts. Libraries A and B were constructed from a cell line obtained from the American
Type Culture Collection. The cell line was derived froma deceased male individual whose body was donated forresearch and whose name was held in con®dence. Library C was made from a donor who signed informed
consent; however, the donor's identity was not anonymous. The D library was constructed from an anonymous donor in accordance with the NIH/DOE Guidelines. For all four library construction, we received
approval from the Caltech Institutional Review Board(IRB).
Donor con®dentiality
When large scale sequencing began at various centers, there was growing concern about maintaining
con®dentiality of the DNA donors from which BAC
DNA libraries were constructed, and also the sequences were obtained. As a result the NHGRI-DOE
Guidance was issued in 1996. In response to the Guidance, we obtained approvals from both the Caltech IRB
and NIH-DOE with regard to the informed consent
form and the sperm collection protocol. The approved
process was utilized to construct library D.
Before donors were asked to provide samples ofblood and semen for the construction of BAC libraries,
they were fully informed about the nature of the experiment and were subsequently asked to sign the informed
consent form. The form fully describes the bene®ts andpotential problems of participating in the program. It
starts by describing how donor con®dentiality will bemaintained, what will be done with the samples, why weneed samples, and who will use samples. The form goeson to explain the bene®ts of participating in the program; e.g., the donor's contribution to the advancementof human health and human genetics. At the same time,
it stresses that the donor will not receive ®nancial gain
and also describes risks of the program; for example, if
the donor's DNA sequence becomes known, the donors
and their families may inadvertently discover the risk ofan illness that had not been known previously. There
would also be the added problem that if the informationis retrieved by employers or insurance companies, it may
become dif®cult to procure a job or health insurance.
In order to reduce the possibility that the identity ofthe donor would become revealed, we implemented thefollowing protocol. To receive samples, we contacted asperm bank and designated one person to contact
donors. Donors who expressed interest in participatingin the project came to the contact person without providing their names, addresses, phone numbers, and anyother identi®able information. The contact person
explained the nature of the human genome project andthe potential bene®ts and risks associated with the
project. After donating blood and semen samples, thedonors were thereafter represented by a randomlychosen four-digit number (even during future semen
donations). Twenty persons served as donors. The semen samples were shipped to Caltech and recorded byalphabetization. The blood samples were shipped to
MIT and used to establish permanent lymphocyte celllines. From the twenty semen samples, we randomly
chose one sperm sample for BAC library construction.
In this way, neither the contact person nor we knew theidentity of the donor since the contact person did notknow the identity of the alphabetized samples and since
we did not know who participated in the program.
Thus, no person, including the donors, knew who had
been chosen for the construction of the BAC library.
Current Applications of the BAC System
BACs are currently used in a wide array of applications, from genome sequencing to gene discovery.
Below are several of the most notable applications to
date.
Genome sequencing
BACs are the major driving force in the effort tosequence the human genome. The collaborative effort
to sequence human chromosome 22 was aided in partby chromosome 22 BAC sub-libraries, which weremade by screening human BAC libraries with sequencetagged site (STS) markers known to be located on
chromosome 22.8±9 By using chromosome 22 BAC sublibraries in addition to cosmid, Fosmid, and P1-derived
arti®cial chromosome (PAC) libraries, the consortiumKeio J Med 2001; 50 (1): 26±30

was able to cover chromosome 22q in 11 clone contigs
with 10 gaps, spanning nearly 33.4 megabases of sequence.10 Recently, the human chromosome 21 has
also been sequenced using BAC maps.11 The relatively
short DNA insert sizes (100±300 kb) and structural
stability make BACs the ideal substrates for direct
sequencing. Additionally, the use of primers based on
pBeloBAC and pBAC108 vectors can be used toquickly and ef®ciently sequence BAC ends, a fact that
greatly facilitates sequencing efforts. For these reasons,
BACs are currently being used in large-scale projects tosequence the human and mouse genomes.
BAC contig assembly
The assembly of BAC contigs remains a critical stepin the formation of physical maps. Classically, building
contigs has involved utilizing restriction endonuclease
digestion to determine pairwise overlap between BAC
clones. Recently, we have described a novel method for
BAC contig assembly which involves multiplexed ¯uorescence-labeled ®ngerprinting.12 In this method, each
BAC clone is digested with three pairs of restriction
endonucleases and labeled with three distinct ¯uorescent dyes, then analyzed on a DNA sequencing machine. By analyzing the ®ngerprinting patterns of two or
more BACs, it is possible to determine overlap among
the BACs and thus develop contigs.
Positional cloning
BACs have been widely used to identify disease locias well as individual genes. One method of identifying
genes involves using positional cloning to identify genetic loci responsible for a disorder. The loci can thenbe analyzed for individual genes by creating physicalmaps of the region via BAC contig assembly (seeabove). This method was the basis for ®nding the breast
cancer susceptibility gene BRCA1.13
BAC library screening
Another method of using BACs to identify genes
entails screening BAC libraries for known sequences.
Such a screen can be carried out by PCR analysis of
BAC library pools, such as row and column pools for all
the plates in a library.7 This method was employed tosearch for human pheromone receptor genes using as
template sources human ESTs with homology to mouse
pheromone receptors (data not published). Once agene is isolated onto an individual BAC, FISH or
Radiation Hybrid mapping can be used to map thecytogenetic localization of the BAC and gene.14 The
short insert sizes and puri®cation ef®ciency of BACs
make them ideal for such cytogenetic analysis.

29

BAC transgenic mice
Traditionally, transgenic mice have been created by
microinjecting copies of a gene as linear DNA into mice
fertilized eggs, and then introducing embryos into amouse. The disadvantage of using single gene copies
is that oftentimes upstream and downstream elementsof the gene are not included in the transgene. An
approach to this problem is to introduce transgenes thatincorporate. BACs rather than coding segments; in thisway, distant regulatory elements such as enhancers areincluded with the gene of interest. For example, an 80
kb P1 bacteriophage spanning the human or mouse
apoB gene conferred expression of apoB in the liver
but not the intestines of transgenic mice. The use of
150±200 kb BACs spanning the apoB gene did confer
apoB expression in both the liver and intestines oftransgenic mice, suggesting that intestinal expression of
apoB is controlled by a distant element that was notpresent on the apoB-carrying P1 bacteriophage.15 An
alternative approach to BAC transgenic mice is the invivo complementation test in which BAC transgenes
are incorporated into mutant mice that lack a gene ofinterest. This ``cloning by rescue'' approach was used as
a complement to positional cloning in order to identify
the circadian Clock gene in mice. In the experiment, a
140 kb BAC transgene was shown to completely rescue
the loss of rhythm phenotype found in Clock mutant
mice, suggesting that the clock gene was located on the
BAC.16

BAC microarrays
As the genome project nears completion, the natureof genomics research will shift from the ``one gene, one
hypothesis'' paradigm to the simultaneous analysis of
thousands of genes in a given experimental system. The
driving force for this breakthrough paradigm is the
microarray, a glass microscope slide that carries thousands of discrete, covalently linked DNA sequences.
By using ¯uorescently linked RNA or cDNA samples,
it is possible to pro®le the gene expression patterns
from a cell line or tissue of interest. The creation of
microarrays carrying BACs will be particularly useful,
especially if the sequence and cytogenetic properties of
the BACs are known. In this way it will be possible tolink gene expression pro®les with chromosomal data
and DNA sequences in order to achieve a global understanding of a disorder or genetic abnormality.17

Acknowledgements: We are grateful to Dr. Mel Simon and many
people who worked on this project, and to the US Department ofEnergy and National Institutes of Health for supporting this work.

30

Shizuya H and Kouros-Mehr H: BAC system development and applications

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