Download Full Text PDF - Mary Ann Liebert, Inc. publishers

CLONING AND STEM CELLS
Volume 4, Number 1, 2002
© Mary Ann Liebert, Inc.
Review
Gene Transfer Strategies in Animal Transgenesis
LLUÍS MONTOLIU
ABSTRACT
Position effects in animal transgenesis have prevented the reproducible success and limited
the initial expectations of this technique in many biotechnological projects. Historically, several strategies have been devised to overcome such position effects, including the progressive addition of regulatory elements belonging to the same or to a heterologous expression
domain. An expression domain is thought to contain all regulatory elements that are needed
to specifically control the expression of a given gene in time and space. The lack of profound
knowledge on the chromatin structure of expression domains of biotechnological interest,
such as mammary gland-specific genes, explains why most standard expression vectors have
failed to drive high-level, position-independent, and copy-number–dependent expression of
transgenes in a reproducible manner. In contrast, the application of artificial chromosometype constructs to animal transgenesis usually ensures optimal expression levels. YACs, BACs,
and PACs have become crucial tools in animal transgenesis, allowing the inclusion of distant
key regulatory sequences, previously unknown, that are characteristic for each expression domain. These elements contribute to insulating the artificial chromosome-type constructs from
chromosomal position effects and are fundamental in order to guarantee the correct expression of transgenes.
genes integrated randomly (Giraldo and Montoliu, 2001).
Genes are thought to be organized on chromosomes as contiguous but independent units
known as expression domains (Elgin, 1990;
Laemmli et al., 1992; Dillon and Grosveld, 1994).
These expression domains are believed to remain
insulated from neighboring sequences and are
thought to include all regulatory elements that
are necessary for their correct gene expression
(Fig. 1). They are frequently represented as chromatin fibers in the form of loops whose ends remain attached to nuclear structural components,
thereby preventing the intercommunication be-
INTRODUCTION
A
TRANSGENESIS has been applied to
study gene function, to generate animal
models of human diseases, or to produce recombinant proteins in milk, among other uses. However, host sequences surrounding the place of
transgene integration can alter the expected expression pattern, turning it ectopic or not detectable. These undesirable events are known as
chromosomal position effects (Wilson et al.,
1990). Several strategies have been devised to
overcome these position effects and thus increase
the probability of optimal expression for transNIMAL
Centro Nacional de Biotecnología (CNB-CSIC), Department of Molecular and Cellular Biology, Madrid, Spain.
39
40
MONTOLIU
FIG. 1. Schematic representation of an expression domain, shielded by two boundaries and including all relevant
regulatory elements that are needed for its correct function.
tween adjacent domains (Bell et al., 2001). The
presence of such boundary elements, at either end
of a given expression domain, has been recently
recognized and identified as one of the most crucial and versatile regulatory elements of a gene
(Bell et al., 2001). An alternative and interesting
interpretation for these important regulatory sequences has also been proposed: boundaries
could be, essentially, elements that would interrupt interactions between enhancers and promoters. As a result of this situation, they would
have been selected against if they occurred within
a given expression domain, but not at the limits
of it. As a consequence, insulators would often be
found at the borders of expression domains,
where the interference with regulatory elements
belonging to the locus is most limited, without
necessarily being selected to function as boundaries (Dillon and Sabatini, 2000).
Two features are normally associated with
these boundary elements. First, they have the
ability to protect against chromosomal position
effects (Kellum and Schedl, 1991). Second, they
can act as positional enhancer blockers interrupting the activation of a promoter by an enhancer (Kellum and Schedl, 1992). A limited number of insulators have been functionally identified
in both vertebrate and invertebrate animal
species in different genes with diverse cell-type
specificity and function (Bell and Felsenfeld,
1999). The addition of heterologous boundaries
has been evaluated in standard transgenesis,
with convincing results in some cases (TaboitDameron et al., 1999).
It is not unexpected that standard transgenes,
usually prepared with limited amount of regulatory sequences from a given expression domain,
might display position effects. Thus, most of the
strategies devised to overcome such position effects have focused their objective on the progressive addition of regulatory elements, aiming to
improve the expression of transgenes. In theory,
if it would be possible to include in a transgenic
construct all regulatory elements that are relevant
for a given expression domain, this strategy
would ensure optimal expression levels irrespective of host sequences surrounding the integration site. Artificial chromosome-type vectors fulfill these criteria, since they are able to incorporate
large genomic segments and include three types
of vectors: yeast artificial chromosomes (YACs),
bacterial artificial chromosomes (BACs), and P1derived artificial chromosomes (PACs; Monaco
and Larin, 1994). It should be noted that artificial
chromosome-type transgenes, irrespective of
their large heterologous DNA insert size, do integrate randomly in the host genome and are not
maintained as episomal chromosomes in mammalian cells (Montoliu et al., 1993). The vector
chromosomal elements included in YACs/
BACs/PACs (centromeres, telomeres, replication
origins) are only functional in their corresponding cell hosts, namely yeast or bacteria for YACs
and BAC/PACs, respectively, and therefore they
are normally not operative in mammalian cells
(Monaco and Larin, 1994). The forthcoming generation of artificial chromosomes, known as
MACs (mammalian artificial chromosomes), are
GENE TRANSFER STRATEGIES IN ANIMAL TRANSGENESIS
expected to incorporate all the benefits of classical artificial chromosome-type vectors with a normal chromosomal maintenance within the mammalian host cells (Monaco and Larin, 1994;
Grimes and Cooke, 1998).
The use of artificial chromosome-type vectors
in transgenesis ensures position-independent,
copy-number–dependent, and optimal levels of
expression of the transgenes, provided all regulatory sequences needed for the establishment
and maintenance of the expression domain (potentially including also the above mentioned
boundary elements) would be located within the
artificial chromosome (Camper and Saunders,
2000; Giraldo and Montoliu, 2001). Thus, artificial
chromosome-type transgenes can circumvent
most position effects observed with standard constructs and are the recommended method of
choice when important regulatory elements from
a given expression domain have not been yet
identified or characterized.
THE MOUSE TYROSINASE EXPRESSION
DOMAIN IN TRANSGENESIS
In our laboratory, we use the mouse tyrosinase
locus as an experimental system to study mammalian expression domains and its behavior in
transgenic animals (Giraldo et al., 1999). Tyrosinase is the key enzyme of melanin synthesis. The
gene is tightly regulated during development and
is expressed in melanocytes, which are derived
from the neural crest, and the retinal pigment epithelium (RPE) cells, which are derived from the
optic cup (Beermann et al., 1992). Mutations
within the tyrosinase gene inactivating its function result in oculocutaneous albinism type I, the
most common type of albinism. In mice, the classical albino mutation corresponds to a single
point mutation in the first exon of the tyrosinase
gene leading to the accumulation of a nonfunctional protein (Jackson and Bennett, 1990). The albino phenotype has been corrected in mice and
other vertebrates, expressing functional tyrosinase transgenes (Beermann et al., 1990; Brem et
al., 1996; Hyodo-Taguchi et al., 1997). However,
standard tyrosinase constructs driven by limited
amounts of regulatory sequences suffer from position effects, display variability in pigmentation,
and usually do not reach wild-type level of expression (Beermann et al., 1990, 1992; Tanaka et
41
al., 1990; Klüppel et al., 1991; Ganss et al., 1994;
Porter et al., 1994).
In contrast, the generation of transgenic mice
with yeast artificial chromosomes (YACs) covering the whole mouse tyrosinase locus totally rescues the hypopigmented phenotype and the retinal abnormalities associated with albinism
(Schedl et al., 1993; Jeffery et al., 1994, 1997). These
results clearly indicated the existence of important regulatory elements, absent in previous standard constructs, which allow YAC tyrosinase
transgenes to overcome common position effects.
One of these regulatory elements, identified
during the characterization of the molecular basis of the chinchilla-mottled (cm ) allele of the albino
locus, was found at 12 kb upstream of the tyrosinase gene (Porter et al., 1991). It was shown
to contain a hypersensitive (HS) DNase I site,
which operated as a cell-specific enhancer in vitro
and in vivo (Ganss et al., 1994; Porter et al., 1994).
This element was evaluated in vivo by generating
specific deletions within YAC tyrosinase transgenes, resulting in the description of a novel locus control region (LCR) at 212 kb in the mouse
tyrosinase gene (Montoliu et al., 1996). The albino
phenotype was completely rescued in animals
whose YAC transgenes kept the LCR. In contrast,
pigmentation was much weaker in transgenic
mice in which the LCR was deleted or substituted
(Montoliu et al., 1996). Recently, we have further
demonstrated that the absence of this key regulatory element of the mouse tyrosinase gene results in variegated expression and delayed retinal pigmentation in transgenic mice (Gimenez et
al., 2001). Our data reveal an important role of
the tyrosinase LCR in establishing the faithful expression pattern of this gene. This is also an illustrative example of how artificial chromosometype transgenes serve to identify distant key
regulatory elements that are characteristic of a
given expression domain.
ARTIFICIAL CHROMOSOME-TYPE
VECTORS CARRYING MAMMARY
GLAND-SPECIFIC GENES
The generation of transgenic animals with artificial chromosome-type vectors has been extended to other mammals with similar success to
that achieved previously with rodents (Giraldo
and Montoliu, 2001). Soon after the initial success
42
MONTOLIU
with mice, transgenic pigs were generated for
xenotransplantation purposes using a YAC with
the entire human membrane–cofactor protein
(MCP) gene (Yannoutsos et al., 1995). Next, rabbits (Brem et al., 1996) and rats (Fujiwara et al.,
1997) were also shown to be useful for the generation and analysis of transgenic animals with
YAC transgenes. In livestock, the benefits of artificial chromosome-type transgenes are expected,
fundamentally, in two fields: the already mentioned xenotransplantation projects and the efficient production of recombinant proteins of interest in the milk of transgenic animals through
the use of mammary gland–specific genes (Brem
et al., 1993; Zuelke et al., 1998; Fujiwara et al.,
1999a; Stinnakre et al., 1999; Houdebine, 1994,
2000).
However, despite the obvious benefits that can
be obtained using YACs or BACs/PACs in mammary gland transgenesis, the description and
characterization of large genomic constructs encompassing mammary gland–specific genes and
its required testing by functional means in vivo
has not progressed as rapidly as could be anticipated (Table 1). The casein locus was among the
first mammary gland–specific genes to be cloned
in artificial chromosome-type vectors. Two YACs
were initially described harboring 380- and 435kb mouse DNA inserts containing the murine casein locus (Tomlinson et al., 1996). Another
group, working independently, also reported the
structure of the murine casein locus from the
mapping analysis of several YACs, ranging from
340 to 460 kb (Rijnkels et al., 1997a). The bigger
YAC corresponded to an equivalent isolated
clone reported before (Tomlinson et al., 1996),
which was obtained from the same mouse YAC
library (Larin et al., 1991). These pioneer reports
with artificial chromosome-type vectors carrying
TABLE 1.
mammary gland–specific genes were triggered
by the need to establish the structure and proper
order of all casein genes (a, b, g, d, k) in the locus, which is about 250 kb long in the mouse genome (Rijnkels et al., 1997a). Several BAC genomic clones were also described from the mouse
casein locus and served to order the casein gene
subunits within the locus (George et al., 1997).
The structure of the human casein gene locus has
also been investigated. A set of four overlapping
YAC genomic clones, ranging in size from 420 to
730 kb, established the physical map of this locus
in the human genome (Rijnkels et al., 1997c).
All this data on mammalian casein gene loci
permitted comparative and evolutionary analyses among these genes (and their structures) in
the mouse, human, and bovine genome (Rijnkels
et al., 1997a,b,c). In contrast, few experiments
have been reported using these large constructs
in transgenic animals. Standard transgenic constructs with promoters from casein genes have
been used with variable success (Brem et al., 1993,
1994; Platenburg et al., 1994; Rijnkels et al. 1997d;
Coulibaly et al., 1999; Houdebine et al., 1994;
2000). Remarkable expression levels of bovine
aS1-casein gene had been reported before in the
milk of transgenic mice using about 14 kb of 59
flanking regions (Rijnkels et al., 1997d). Highlevel expression of heterologous recombinant
proteins has been also achieved with smaller, 2.9kb (Brem et al., 1994; Coulibaly et al., 1999) or 8kb 59 promoter regions of the bovine aS1-casein
gene (Platenburg et al., 1994). However, in all
these cases, expression levels were highly variable and integration-site dependent, thus suggesting that not all cis-regulatory elements involved in the control of bovine aS1-casein gene
expression were included in these constructs. The
use of artificial chromosome-type vectors har-
ARTIFICIAL CHROMOSOME -TYPE VECTORS CARRYING MAMMARY GLAND –SPECIFIC GENES
Gene
Type
Size (kb)
Reference
Murine casein locus
Murine casein locus
Murine casein locus
Human casein locus
Human a-lactalbumin
Bovine casein locus
Goat a-lactalbumin
Porcine WAP
Murine WAP
Murine WAP
YAC
BAC
YAC
YAC
YAC
BAC
YAC
BAC
BAC
YAC
380, 435
105, 155
340, 370, 460
420, 520, 700, 730
210
100
160
125, 145
20, 25
~300
Tomlinson et al (1996)
George et al. (1997)
Rijnkels et al. (1997a)
Rijnkels et al. (1997c)
Fujiwara et al. (1997)
Zuelke et al. (1999)
Stinnakre et al. (1999)
Rival et al. (2001)
Aguirre et al. (unpublished observations)
Aguirre and Montoliu (unpublished observations)
GENE TRANSFER STRATEGIES IN ANIMAL TRANSGENESIS
boring larger genomic pieces of casein gene flanking regulatory regions appears as a potential
solution for this problem and should be investigated. In this regard, a BAC covering part of the
mouse casein gene locus (100-kb insert) was recently tested in transgenic mice and showed highlevel expression (Zuelke et al., 1999).
With the same aim, namely, trying to overcome
the usual suboptimal performance of classical
plasmid-based mammary gland–specific transgenes, several additional artificial chromosometype vectors have been isolated and tested in
transgenic animals. A 210-kb YAC carrying the
human a-lactalbumin gene was shown to drive
position-independent and high-level expression
in transgenic rats (Fujiwara et al., 1997). The same
laboratory (Fujiwara et al., 1999a) reported the
use of this 210-kb YAC to produce high levels of
human growth hormone (hGH) in the milk of
transgenic rats, by inserting the hGH gene using
homologous recombination techniques in yeast.
Additionally, Fujiwara et al. (1999b) extended the
search and analysis of distant regulatory elements of the human a-lactalbumin locus using
new engineered constructs derived from the original 210-kb YAC. Similarly, a 170-kb BAC encompassing the homologous goat a-lactalbumin
gene was isolated and reported to display highlevel, position-independent, and copy number–
related expression in transgenic mice (Stinnakre
et al., 1999).
The gene encoding the whey acidic protein
(WAP), the major whey protein in rodents, rabbits, and camel, has been used in most mammary
gland–specific transgenic experiments. Standard
WAP expression vectors have been routinely
transferred to the germ line of animals and shown
43
to drive mammary gland–specific expression of
heterologous genes (Burdon et al., 1991; Bischoff
et al., 1992; Li and Rosen, 1994; Castilla et al.,
1998). However, very soon, investigators realized
that standard short WAP constructs, with a limited number of regulatory sequences, were not
capable of overcoming position effects and thus
heterologous genes were found ectopically expressed or produced at very low levels (Limonta
et al., 1995; Rosen et al., 1996; Massaud et al., 1996;
Aguirre et al., 1998). More distal regulatory elements were identified in the rat WAP gene (Krnacik et al., 1995; Li and Rosen, 1994) and in the
rabbit WAP gene (Bischoff et al., 1992; Devinoy
et al., 1994), and were shown to partially compensate the suboptimal expression capabilities in
transgenic animals. Alternatively, the combined
use of standard WAP sequences and heterologous
scaffold/matrix attachment regions (S/MARs)
in transgenic animals has proven to impart position effects in some cases (McKnight et al., 1992,
1996).
The WAP gene also exists in the porcine genome, and it is regulated like its homologous
counterparts in rodents (Simpson et al., 1998). Recently, several BAC clones have been isolated and
characterized harboring the porcine WAP gene in
genomic fragments as large as 145 kb (Rival et al.,
2001). The functional analysis of these porcine
BAC WAP constructs in transgenic animals will
clarify their potential as reproducible and efficient mammary gland–specific expression vectors.
In our laboratory, we have isolated two BACs
(20- and 25-kb inserts) containing genomic sequences of the mouse WAP locus (A. Aguirre, A.
Lavado, L. Regales, P. Giraldo, and L. Montoliu,
FIG. 2. Construct size matters in animal transgenesis. Three hypothetical overlapping artificial chromosome-type
vectors are shown (constructs A, B, and C). The bigger construct (C) harbors all DNase I hypersensitive sites (vertical arrows) that are associated with this expression domain, usually identifying regulatory elements. Construct C is
expected to faithfully reproduce the expression pattern of the gene in ectopical genomic locations.
44
MONTOLIU
unpublished observations). To evaluate the potential of these mouse BAC WAP constructs as
mammary gland–specific expression vectors, we
have inserted, in frame, as a transcriptional fusion, a reporter gene (secreted alkaline phosphatase, SEAP) by ET-cloning/GET recombination
techniques, a recently pioneered methodology
that use alternative recombination enzymatic
pathways to allow targeted modification of
BAC/PAC constructs by homologous recombination in bacterial cells (Zhang et al., 1998;
Narayanan et al., 1999; reviewed in Muyrers et
al., 2001). Experiments are currently in progress
to determine the performance of these recombinant mouse BAC WAP transgenes in vivo, compared with standard WAP-SEAP transgenic mice.
In parallel, we have isolated and preliminary
characterized a 300-kb YAC encompassing the
mouse WAP locus that we plan to test in transgenic experiments (A. Aguirre and L. Montoliu,
unpublished observations).
CONCLUSION
The use of YAC, BAC, and PAC constructs is
usually associated with optimal performance in
transgenic experiments. The size of their genomic
inserts habitually ensures the inclusion of most
regulatory elements that are relevant for the right
expression of a given gene. Therefore, artificial
chromosome-type transgenes are normally expressed in correct spatial- and temporal-specific
manners (Giraldo and Montoliu, 2001).
The size of heterologous DNA inserts in artificial chromosome-type vectors does matter because it increases the probability of encompassing all regulatory elements (known or unknown)
that are important for the correct expression of
the gene and constitute the expression domain.
However, what really matters for the most efficient performance of these large vectors in animal transgenesis is the presence of such distant
regulatory elements (including boundaries or insulators) within the constructs, irrespective of
how close or far they could be from the body of
the gene (Fig. 2). Thus, the abnormal expression
patterns occasionally observed with some artificial chromosome-type of transgenes can be subsequently explained by the absence of additional
regulatory elements that constitute a given expression domain. Once these additional elements
are incorporated in larger constructs, faithful ex-
pression pattern of the transgene is obtained
(Lakshmanan et al., 1998, 1999).
The benefits of artificial chromosome transgenesis will soon be exported to biotechnological
applications and, in some cases, have already
been foreseen (Fujiwara et al., 1997; Zuelke, 1998,
1999; Stinnakre et al., 1999; Rival et al., 2001).
These benefits will mostly include the production
of recombinant proteins of interest in the mammary gland of transgenic animals, with the hope
that animal transgenesis will eventually become
more reproducible, efficient, and predictable.
ACKNOWLEDGMENTS
This work has been supported by funds from
CAM, CICYT (Plan Nacional I1D), AECI, and
Laboratorios Dr. Esteve, S.A.
REFERENCES
Aguirre, A., Castro-Palomino, N., De la Fuente, J., et al.
(1998). Expression of human erythropoietin transgenes
and of the endogenous WAP gene in the mammary
gland of transgenic rabbits during gestation and lactation. Transgenic Res. 7, 311–317.
Beermann, F., Ruppert, S., Hummler, E., et al. (1990). Rescue of the albino phenotype by introduction of a functional tyrosinase gene into mice. EMBO J. 9, 2819–2826.
Beermann, F., Schmid, E., and Schütz, G. (1992). Expression of the mouse tyrosinase gene during embryonic
development: recapitulation of the temporal regulation
in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 89,
2809–2813.
Bell, A.C., and Felsenfeld, G. (1999). Stopped at the border: boundaries and insulators. Curr. Opin. Genet. Dev.
9, 191–198.
Bell, A.C., West, A.G., and Felsenfeld, G. (2001). Insulators and boundaries: versatile regulatory elements in
the eukaryotic genome. Science 291, 447–450.
Bischoff, R., Degryse, E., Perraud, F., et al. (1992). A 17.6kbp region located upstream of the rabbit WAP gene
directs high level expression of a functional human protein variant in transgenic mouse milk. FEBS Lett. 305,
265–268.
Brem, G., Besenfelder, U., and Hartl, P. (1993). Production of foreign proteins in the mammary glands of
transgenic animals. Chim. Oggi May, 21–25.
Brem, G., Hartl, P., Besenfelder, U., et al. (1994). Expression of synthetic cDNA sequences encoding human insulin-like growth factor–I (IGF-I) in the mammary
gland of transgenic rabbits. Gene 149, 351–355.
Brem, G., Besenfelder, U., Aigner, B., et al. (1996). YAC
transgenesis in farm animals: rescue of albinism in rabbits. Mol. Reprod. Dev. 44, 56–62.
Burdon, T., Sankaran, L., Wall, R.J., et al. (1991). Expres-
GENE TRANSFER STRATEGIES IN ANIMAL TRANSGENESIS
sion of a whey acidic protein transgene during mammary development. J. Biol. Chem. 266, 6909–6914.
Camper, S.A., and Saunders, T.L. (2000). Transgenic rescue of mutant phenotypes using large DNA fragments.
In Genetic Manipulation of Receptor Expression and Function. Accili, D., ed. (John Wiley and Sons, New York)
pp 1–22.
Castilla, J., Pintado, B., Sola, I., et al. (1998). Engineering
passive immunity in transgenic mice secreting virusneutralizing antibodies in milk. Nat. Biotechnol. 16,
349–354.
Coulibaly, S., Besenfelder, U., Fleischmann, M., et al.
(1999). Human nerve growth factor–beta (hNGF-b):
mammary gland specific expression and production in
transgenic rabbits. FEBS Lett. 444, 111–116.
Dillon, N., and Grosveld, F. (1994). Chromatin domains
as potential units of eukaryotic gene function. Curr.
Opin. Genet. Dev. 4, 260–264.
Dillon, N., and Sabbatini, P. (2000). Functional gene expression domains: defining the functional unit of eukaryotic gene regulation. BioEssays 22, 657–665.
Devinoy, E., Thepot, D., Stinnakre, M.G., et al. (1994).
High-level production of human growth hormone in
the milk of transgenic mice: the upstream region of the
rabbit whey acidic protein (WAP) gene targets transgene expression to the mammary gland. Transgenic
Res. 3, 79–89.
Elgin, S.C.R. (1990). Chromatin structure and gene activity. Curr. Opin. Cell. Biol. 2, 437–445.
Fujiwara, Y., Miwa, M., Takahashi, R., et al. (1997). Position-independent and high-level expression of human
alpha-lactalbumin in the milk of transgenic rats carrying a 210-kb YAC DNA. Mol. Reprod. Dev. 47, 157–163.
Fujiwara, Y., Miwa, M., Takahashi, R., et al. (1999a). Highlevel expressing YAC vector for transgenic animal
bioreactors. Mol. Reprod. Dev. 52, 414–420.
Fujiwara, Y., Takahashi, R.I., Miwa, M., et al. (1999b).
Analysis of control elements for position-independent
expression of human alpha-lactalbumin YAC. Mol. Reprod. Dev. 54, 17–23.
Ganss, R., Montoliu, L., Monaghan, A.P., et al. (1994). A
cell-specific enhancer far upstream of the mouse tyrosinase gene confers high level and copy number-related expression in transgenic mice. EMBO J. 13, 3083–
3093.
George, S., Clark, A.J., and Archibald, A.L. (1997). Physical mapping of the murine casein locus reveals the
gene order as a–b–g–e –k. DNA Cell Biol. 16, 477–484.
Gimenez, E., Giraldo, P., Jeffery, G., et al. (2001). Variegated expression and delayed retinal pigmentation during development in transgenic mice with a deletion in
the locus control region of the tyrosinase gene. Genesis 30, 21–25.
Giraldo, P., Gimenez, E., and Montoliu, L. (1999). The use
of yeast artificial chromosomes in transgenic animals:
expression studies of the tyrosinase gene in transgenic
mice. J. Genet Anal. 15, 175–178.
Giraldo, P., and Montoliu, L. (2001). Size matters: use of
YACs, BACs and PACs in transgenic animals. Transgenic Res. 10, 83–103.
45
Grimes, B., and Cooke, H. (1998). Engineering mammalian chromosomes. Hum. Mol. Genet. 7, 1635–1640.
Houdebine, L.M. (1994). Production of pharmaceutical
proteins from transgenic animals. J. Biotechnol. 34, 269–
287.
Houdebine, L.M. (2000). Transgenic animal bioreactors.
Transgenic Res. 9, 305–320.
Hyodo-Taguchi, Y., Winkler, C., Kurihara, Y., et al. (1997).
Phenotypic rescue of the albino mutation in the medakafish (Oryzias latipes) by a mouse tyrosinase transgene.
Mech. Dev. 68, 27–35.
Jackson, I.J., and Bennett, D.C. (1990). Identification of the
albino mutation of mouse tyrosinase by analysis of an
in vitro revertant. Proc. Natl. Acad. Sci. U.S.A. 87, 7010–
7014.
Jeffery, G., Schutz, G., and Montoliu, L. (1994). Correction
of abnormal retinal pathways found with albinism by
introduction of a functional tyrosinase gene in transgenic mice. Dev. Biol. 166, 460–464.
Jeffery, G., Brem, G., and Montoliu, L. (1997). Correction
of retinal abnormalities found in albinism by introduction of a functional tyrosinase gene in transgenic mice
and rabbits. Dev. Brain Res. 99, 95–102.
Kellum, R., and Schedl, P. (1991). A position-effect assay
for boundaries of higher order chromosomal domains.
Cell 64, 941–950.
Kellum, R., and Schedl, P. (1992). A group of scs elements
function as domain boundaries in an enhancer-blocking assay. Mol. Cell. Biol. 12, 2424–2431.
Kluppel, M., Beermann, F., Ruppert, S., et al. (1991). The
mouse tyrosinase promoter is sufficient for expression
in melanocytes and in the pigmented epithelium of the
retina. Proc. Natl. Acad. Sci. U.S.A. 88, 3777–3781.
Krnacik, M.J., Li, S., Liao, J., et al. (1995). Position-independent expression of whey acidic protein transgenes.
J. Biol. Chem. 270, 11119–11129.
Laemmli, U.K., Käs, E., Poljak, L., et al. (1992). Scaffoldassociated regions: cis-acting determinants of chromatin structural loops and functional domains. Curr.
Opin. Genet. Dev. 2, 275–285.
Lakshmanan, G., Lieuw, K.H., Grosveld, F., et al. (1998).
Partial rescue of GATA-3 by yeast artificial chromosome transgenes. Dev. Biol. 204, 451–463.
Lakshmanan, G., Lieuw, K.H., Lim, K.C., et al. (1999). Localization of distant urogenital system–, central nervous system–, and endocardium-specific transcriptional regulatory elements in the GATA-3 locus. Mol.
Cell. Biol. 19, 1558–1568.
Larin, Z., Monaco, A.P., and Lehrach, H. (1991). Yeast artificial chromosome libraries containing large inserts
from mouse and human DNA. Proc. Natl. Acad. Sci.
U.S.A. 88, 4123–4127.
Li, S., and Rosen, J.M. (1994). Distal regulatory elements
required for rat whey acidic protein gene expression in
transgenic mice. J. Biol. Chem. 269, 14235–14243.
Limonta, J.M., Castro, F.O., Martinez, R., et al. (1995).
Transgenic rabbits as bioreactors for the production of
human growth hormone. J. Biotechnol. 40, 49–58.
Massaud, M., Attal, J., Thepot, D., et al. (1996). The deleterious effects of human erythropoietin gene driven by
46
the rabbit whey acidic protein gene promoter in transgenic rabbits. Reprod. Nutr. Dev. 36, 555–563.
McKnight, R.A., Shamay, A., Sankaran, L., et al. (1992).
Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 89, 6943–6947.
McKnight, R.A., Spencer, M., Wall, R.J., et al. (1996). Severe position effects imposed on a 1-kb mouse whey
acidic protein gene promoter are overcome by heterologous matrix attachment regions. Mol. Reprod. Dev. 44,
179–184.
Monaco, A.P., and Larin, Z. (1994). YACs, BACs, PACs
and MACs: artificial chromosomes as research tools.
Trends Biotechnol. 12, 280–286.
Montoliu, L., Schedl, A., Kelsey, G., et al. (1993). Generation of transgenic mice with yeast artificial chromosomes. Cold Spring Harb. Symp. Quant. Biol. 58, 55–62.
Montoliu, L., Umland, T., and Schutz, G. (1996). A locus
control region at 212 kb of the tyrosinase gene. EMBO
J. 15, 6026–6034.
Muyrers, J.P.P., Zhang, Y., and Stewart, A.F. (2001). Techniques: recombinogenic engineering— new options for
cloning and manipulating DNA. Trends Biochem. Sci.
26, 325–331.
Narayanan, K., Williamson, R., Zhang, Y., et al. (1999). Efficient and precise engineering of a 200-kb beta-globin
human/bacterial artificial chromosome in E. coli
DH10B using an inducible homologous recombination
system. Gene Ther. 6, 442–447.
Platenburg, G.J., Kootwijk, E.P.A., Kooiman, P.M., et al.
(1994). Expression of human lactoferrin in milk of transgenic mice. Transgenic Res. 3, 99–108.
Porter, S., Larue, L., and Mintz, B. (1991). Mosaicism of
tyrosinase-locus transcription & chromatin structure in
dark vs. light melanocyte clones of homozygous chinchilla-mottled mice. Dev. Genet. 12, 393–402.
Porter, S.D., and Meyer, C.J. (1994). A distal tyrosinase
upstream element stimulates gene expression in neuralcrest–derived melanocytes of transgenic mice: positionindependent and mosaic expression. Development 120,
2103–2111.
Rijnkels, M., Wheeler, D.A., de Boer, H.A., et al. (1997a).
Structure and expression of the mouse casein gene locus. Mamm. Genome 8, 9–15.
Rijnkels, M., Kooiman, P.M., de Boer, H.A., et al. (1997b).
Organization of the bovine casein gene locus. Mamm.
Genome 8, 148–152.
Rijnkels, M., Meerschoek, E., de Boer, H.A., et al. (1997c).
Physical map and localization of the human casein gene
locus. Mamm. Genome 8, 285–286.
Rijnkels, M., Kooiman, P.M., Platenburg, G.J., et al.
(1997d). High-level expression of bovine S1-casein in
milk of transgenic mice. Transgenic Res. 7, 5–14.
Rival, S., Attal, J., Delville-Giraud, C., et al. (2001).
Cloning, transcription and chromosomal localization of
the porcine whey acidic protein gene and its expression
in HC11 cell line. Gene 267, 37–47.
MONTOLIU
Rosen, J.M., Li, S., Raught, B., et al. (1996). The mammary
gland as a bioreactor: factors regulating the efficient expression of milk protein–based transgenes. Am. J. Clin.
Nutr. 63, 627S–632S.
Schedl, A., Montoliu, L., Kelsey, G., et al. (1993). A yeast
artificial chromosome covering the tyrosinase gene confers copy number–dependent expression in transgenic
mice. Nature 362, 258–261.
Simpson, K.J., Bird, P., Shaw, D., et al. (1998). Molecular
characterisation and hormone-dependent expression of
the porcine whey acidic protein gene. J. Mol. Endocrinol. 20, 27–35.
Stinnakre, M.G., Soulier, S., Schibler, L., et al. (1999). Position-independent and copy-number–related expression of a goat bacterial artificial chromosome alpha-lactalbumin gene in transgenic mice. Biochem J. 339, 33–
36.
Taboit-Dameron, F., Malassagne, B., Viglietta, C., et al.
(1999). Association of the 59HS4 sequence of the chicken
beta-globin locus control region with human EF1 alpha
gene promoter induces ubiquitous and high expression
of human CD55 and CD59 cDNAs in transgenic rabbits. Transgenic Res. 8, 223–235.
Tanaka, S., Yamamoto, H., Takeuchi, S., et al. (1990).
Melanization in albino mice transformed by introducing cloned mouse tyrosinase gene. Development 108,
223–227.
Tomlinson, A.M., Cox, R.D., Lehrach, H.R., et al. (1996).
Restriction map of two yeast artificial chromosomes
spanning the murine casein locus. Mamm. Genome 7,
542–544.
Wilson, C., Bellen, H.J., and Gehring, W.J. (1990). Position
effects on eukaryotic gene expression. Annu. Rev. Cell.
Biol. 6, 679–714.
Yannoutsos, N., Langford, G.A., Cozzi, E., et al. (1995).
Production of pigs transgenic for human regulators of
complement activation. Transplant. Proc. 27, 324–325.
Zhang, Y., Buchholz, F., Muyrers, J.P., et al. (1998). A new
logic for DNA engineering using recombination in
Escherichia coli. Nat. Genet. 20, 123–128.
Zuelke, K.A. (1998). Transgenic modification of cows milk
for value-added processing. Reprod. Fertil. Dev. 10,
671–676.
Zuelke, K.A., Walker, C., Price, K., et al. (1999). Expression of a 100-kb bacterial artificial chromosome (BAC)
genomic clone of bovine aS1-casein in transgenic mice.
Therogeniology 51, 430.
Address reprint requests to:
Dr. Lluís Montoliu
Centro Nacional de Biotecnologia (CNB-CSIC)
Department of Molecular and Cellular Biology
Campus de Cantoblanco
28049 Madrid, Spain
E-mail: [email protected]