Construction and characterization of a porcine P1-derived artificial
chromosome (PAC) library covering 3.2 genome equivalents and
cytogenetical assignment of six type I and type II loci
Hiam K. Al-Bayati,
Institute of Veterinary Medicine, University of Go¨ttingen, Groner Landstrasse 2 37073 Goettingen, Germany
Institute of Animal Breeding, Technical University of Munich/Weihenstephan, Germany
Medical Laboratory for Human Genetics Dr. Mehnert, Neu-Ulm, Germany
Received: 24 November 1998 / Accepted: 1 February 1999
Abstract. A porcine P1-derived artificial chromosome (PAC) li-
brary of a male German Landrace pig was constructed in
pCYPAC2. In total 90,240 clones were generated and individually
transferred into microtiter plates. An average insert size of 119.1
kb was determined by analyzing 150 randomly selected PAC
clones by pulsed field electrophoresis, yielding approximately 3.2
genome equivalents. The stability of nine clones was followed
through 110 generations showing no reduction of the insert size.
The probability of identifying a specific chromosomal region
within the library was tested by screening for the presence of seven
type I and five type II loci. The analysis showed that most loci
(10/12) were present in the library at least twice. To determine the
percentage of chimerism, six clones were analyzed by fluorescence
in situ hybridization (FISH) on metaphase chromosomes. We as-
sign one type I locus (Triadin) and three type II loci (SW855,
Compared with human or mouse genome analysis, mapping of
most livestock species is still at the beginning. However, in a few
species, that is, cattle and pig, the density of mapped type I and
type II loci has now reached a stage where the generation of
contigs between two markers for the isolation of linked genes can
be achieved. To accomplish these objectives, it is essential to have
a sufficient number of large insert libraries. Currently, at least two
BAC and four YAC libraries are available for porcine genome
mapping (Alexander et al. 1997; Leeb et al. 1995; Rogel-Gaillard
et al. 1997; Zehetner and Lehrach 1994).
With the development of yeast artificial chromosomes (YACs),
a system became available that allowed the cloning of DNA frag-
ments up to several megabasepairs (Burke et al. 1992; Dausset et
al. 1992; Larin et al. 1991). Although large YACs are advanta-
geous over smaller insert clones for the development of long-range
physical maps, problems have been described with the construc-
tion and handling of YAC libraries and YACs. These problems
are, for example, the low transformation efficiency, the difficulty
to separate and isolate YAC DNA from the yeast chromosomal
background, the genomic instability, and the high proportion of
chimerisms (Bronson et al. 1991; Cai et al. 1995; Green et al.
1991; Larionov et al. 1994; Schmidt et al. 1994; Smith et al. 1990).
However, YACs are still an extremely important tool for long-
range genome mapping.
Efforts have been made to overcome the limitations of YACs
by developing an alternative system using the bacteriophage P1 as
vector (Pierce et al. 1992; Rao et al. 1992; Sternberg et al. 1990).
The P1 system has a number of desirable features, such as the
stability of the inserts and a simple selection of the recombinants
from non-recombinants. However, the cloning capacity of the P1
system is only around 95 kb, and the complicated in vitro pack-
aging requirements limit the applicability.
Another cloning system based on the E. coli F-factor, that is,
bacterial artificial chromosome (BAC), was developed by Shizuya
and associates (1992) with a cloning capacity of up to 300 kb.
Despite the advantages of the BAC cloning system, a drawback is
the low amount of recombinant DNA that can be recovered from
a colony because of the single-copy control of the F-factor
(Koppes 1992; Shizuya et al. 1992).
Recently, a new system combining the desirable features of the
P1 and the BAC systems was described, designated P1-derived
artificial chromosomes (PAC, Ioannou et al. 1994). The PAC sys-
tem, which has a capacity ranging from 100 to 300 kb, is charac-
terized by a high cloning efficiency and insert stability. So far,
almost no chimerism was detected (Ashworth et al. 1995; Hubert
et al. 1997; Ioannou et al. 1994; Matsumoto et al. 1997;
Nechiporuk et al. 1997; Nothwang et al. 1997). The PAC system
has been used to construct chromosome-specific as well as com-
plete human, rat, canine, baboon, parasite, and murine libraries
(Gingrich et al. 1996; Ioannou et al. 1994; Osoegawa et al. 1998;
Piper et al. 1998; Woon et al. 1998). In this report, we describe the
cloning and characterization of a porcine PAC library with a 3.2
coverage of the porcine genome.
Materials and methods
Isolation, partial digestion, and size selection of porcine geno-
High-molecular-weight DNA was prepared from white blood
cells of a healthy male German Landrace pig according to standard pro-
tocols (Ausubel et al. 1995). Briefly, isolated cells were washed with PBS
buffer (pH 8.0) twice, and the concentrations were adjusted to6×10
per ml. An equal volume of 1% low-melting-point agarose (BioRad,
Mu¨nchen) was gently added, and the mixture was incubated at 37°C. The
cell suspension was transferred into pre-cooled 80-l BioRad plug molds.
The blocks were incubated for 20 min at 4°C and extruded directly into 50
ml proteinase K buffer. The blocks were incubated at 50°C for1hina
water bath shaker. The proteinase K buffer was exchanged, and proteinase
K was added (2 mg proteinase K/ml proteinase K buffer). The blocks were
incubated at 50°C overnight and then washed three times with TE buffer at
room temperature (RT). After washing, the blocks were dialyzed twice
against 50 ml TE buffer containing PMSF (40 mg/ml) at 50°C for 30 min
with gentle shaking. Finally, the blocks were washed twice with TE buffer
and stored in TE at 4°C.
For partial digestion, each block was equilibrated in 1 ml of 1 × MboI
restriction enzyme buffer (Biolabs, Schwaebach) at RT for 10 min. After
pre-incubation, the blocks were transferred separately into 120 l 1 × re-
Correspondence to: B. Brenig
Mammalian Genome 10, 569–572 (1999).
© Springer-Verlag New York Inc. 1999