Download Chromosomal insertion of foreign DNA

Chromosomal insertion of foreign DNA
Jo Bishop
To cite this version:
Jo Bishop. Chromosomal insertion of foreign DNA. Reproduction Nutrition Development, EDP
Sciences, 1996, 36 (6), pp.607-618.
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Submitted on 1 Jan 1996
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Review article
Chromosomal insertion of foreign DNA
JO
Centre of Genome Research,
Bishop
University of Edinburgh, King’s Buildings, Edinburgh EH9 3JQ,
(Received
26
UK
July 1996; accepted 9 September 1996)
Summary ― The main route and, in most species, the only reliable route to the generation of transgenic animals is by microinjecting DNA into an early embryo, generally one of the pronuclei of a newly
fertilized egg (a one-cell embryo). In most cases, a small number (perhaps 100) of identical cloned DNA
molecules is introduced in this way. The weight of evidence supports the view that this DNA forms extrachromosomal concatemers (arrays), mainly of monomers orientated in the same direction, by rounds
of homologous recombination. Since this occurs when a population of identical linear molecules is
introduced, productive recombination can only take place after a population of circularly permuted
monomers has been generated by circularization and random cleavage. Extrachromosomal recombination is known to occur by a nonconservative process in transfected mammalian cells in culture. Concatemeric molecules integrate into the chromosomes, more or less at random, by illegitimate recombination. This may occur during DNA replication, consistent with the very high observed frequency of
transgenic founder animals that are mosaics of transgenic and nontransgenic cells. Foreign genes
integrated in this way are frequently liable to chromosomal position effects, which can adversely affect
expression. In the commercial arena this often necessitates the production of a large number of transgenic founders in the hope of obtaining one with a high expression level. Ways of approaching this practical problem are explored.
DNA
microinjection I integration I recombination
Résumé ― Insertion chromosomique d’ADN étranger. La voie principale, et, chez la plupart des
espèces, la seule voie fiable pour l’obtention d’animaux transgéniques, est la micro-injection dADN dans
un jeune embryon, généralement dans un des pronoyaux du zygote. Un petit nombre (une centaine)
de molécules dADN clonées identiques est introduit de cette manière. Les données expérimentales
indiquent que cet ADN forme des concatémères extrachromosomiques composés essentiellement
de monomères orientés dans la même direction à la suite d’un processus de recombinaisons homologues. Puisque cela se produit à partir d’une population de molécules linéaires identiques, une recombinaison homologue ne peut advenir qu’après une circularisation des monomères suivie d’une coupure
au hasard. La recombinaison extrachromosomique est connue pour se produire selon un processus
non conservatif dans des cellules mammaliennes transfectées en culture. Des molécules concatémères sont intégrées dans des chromosomes à peu près au hasard, par recombinaison illégitime.
Cela peut arriver pendant la replication de 1 ADN, et ce fait est compatible avec l’obtention à une
grande fréquence d’animaux fondateurs transgéniques mosaïques pour le transgène. Des gènes
étrangers intégrés de cette manière sont fréquemment dépendants de l’effet position dans les chroqui peut inhiber l’expression. Dans le domaine commercial, cela oblige souvent à produire
un grand nombre de fondateurs transgéniques dans l’espoir d’en obtenir un ayant un niveau d’expression élevé. Les moyens de résoudre ce problème pratique sont abordés.
mosomes
micro-injection d’ADNlmécanisme d’intégration / recombinaison
INTRODUCTION
Transgene integration undoubtedly presents
important opportunities both for scientific
progress and for commercial applications.
On the scientific side there is ample evidence that immortalized cultured cells very
often mimic only very poorly the cells from
which they originated, to the extent that the
expression of resident genes is often quite
unlike that of the same genes in vivo. Similarly, and probably for the same reasons,
the expression of a transgene within transfected cultured cells is often quite unlike
that of the same gene when incorporated
into the genome of a living animal. Available evidence links this unwelcome infidelity
to not unexpected changes in the population of transcription factors that accompany
explantation and immortalization. Thus,
while a great deal may be learned quite
rapidly from the use of transfected cell lines,
the uncomfortable fact is that the picture
that emerges, although an accurate description of the in vitro situation, may be a wholly
inadequate description of the situation in
vivo. Often this discrepancy will only become
apparent when the in vivo situation is
assessed by the transgenic route. Again,
quite obviously the transgenic approach
offers unique opportunities to investigate
systemic effects of a gene, or its effects on
tissue-tissue interactions or on development. In the commercial arena, there are
obvious possibilities for animal improvement
and there have already been commercial
successes in the genetic improvement of
plants (Tabe et al, 1993; Hackland et al,
1994). Similarly, both animals and plants
can be engineered to become producers of
therapeutic proteins (Hoyer
et
al, 1994;
Janne et al, 1994; Cramer et al, 1996).
For many purposes the preferred route to
an altered genome is recombination
between a transgene and homologous resident DNA in totipotent embryonic stem (ES)
cells followed by the introduction of the engineered cells into the inner cell mass of host
blastocysts and germline transmission from
the resulting chimera (Robertson, 1991;
Smith, 1992). A gene altered by this route is
genetically equivalent to a conventional
mutation, differing only in that a chosen gene
has been altered and the specific change
made has been predetermined. To date,
this approach is available only in mice, and
especially in strain 129 mice, because,
despite a considerable effort, ES cell lines
with suitable properties have not been established from other species.
For some purposes homologous recombination is not the most appropriate
approach, even in mice. These include the
introduction of a supernumerary gene with
which there are no resident homologies and
the introduction of genes designed to give
high level ectopic expression without impairing the normal functioning of any resident
gene. The engineering of ’animal bioreactors’ falls into this category. Transgenic animals of these types have overwhelmingly
been produced by directly injecting DNA
into one of the two pronuclei of newly fertilized eggs. Integration into the chromosomes follows with a surprisingly high frequency, making the process of generating
transgenic founders quite efficient. With
most genes, however, expression is problematical: the problems most commonly
encountered are lack of expression, vari-
ability of expression
in different transgenic
lines that carry the same transgene and
expression that is inappropriate in terms of
time (developmental appearance), place
(tissue specificity) or both. In practice, variable expression is the most consistently
troublesome, and thus, in order to obtain a
transgenic line that exhibits an acceptable
level of expression, many transgenic
founders must be generated and analysed,
with obvious cost implications. This will be
the central theme to which other parts of
the present review will be directed.
THE FATE OF DNA IN MICROINJECTED
EMBRYOS AND TRANSFECTED CELLS
There is little or no direct evidence relating
to the integration of foreign DNA into the
chromosomes of microinjected early
embryo nuclei. Instead, there is surmise
based on analogy with transfected or
microinjected cells in culture. The evidence
that justifies this analogy derives from the
similar organization of integrated DNA in
the two situations. The analysis of integrated DNA is necessarily carried out many
cell generations after the initial molecular
events occur, during which time rearrangements may have taken place, and this
needs to be borne in mind. Second, we
may note the corollary that each analysis
is carried out on the products of clonal
growth of a single cell that received the foreign DNA, in one case an embryo and in
the other a single cell that settled in a culture dish. The most telling observation
stems from experiments in which a single
genetically manipulated linear DNA species,
ie, a homogeneous population of identical
linear molecules, is introduced. This is the
common practice in embryo microinjection,
but is relatively rare among studies of DNA
integration in transfected or microinjected
cells. In the latter case, an excess of ‘carrier’
DNA (eg, salmon sperm DNA) has com-
monly been mixed in with the cloned
gene(s), making a meaningful analysis of
integrated foreign DNA impossible. However, in those cases in which carrier DNA
not employed the outcome was remarkably similar to the outcome from microinwas
jection of DNA into one-cell embryos: i) We
find that the DNA commonly integrates into
only one or at most a few chromosomal
sites in a given embryo or cell clone; ii) At
each site multiple copies of the foreign DNA
are commonly integrated together; iii) Most
of the multiple copies at a given site are
arranged in direct (ie, the same) orientation; iv) Most importantly, these direct tandem copies are perfect or near-perfect
copies of the input DNA, with sometimes
minor imperfections at the junctions
between neighbouring copies; v) More
rarely, copies are found in inverted orientation, and these copies sometimes have a
terminal deletion (reviewed in Bishop and
Smith, 1989).
We previously argued that foreign DNA
arrays are generated extrachromosomally
prior to integration into the chromosomes
(Bishop and Smith, 1989). This argument is
based on two considerations, neither of
which is compelling. First, a priori, productive collisions between two extrachromosomal DNA molecules are likely to be more
frequent than those between an extrachromosomal molecule and any that happen already to have become integrated. Inn
addition, extrachromosomal interactions
have been extensively documented in
transfected cells (Lin et at, 1984; Wake et
ai, 1985). Some of the evidence from transfected cells relates to the mechanisms
underlying the formation of extrachromosomal arrays. This tells us that two different
mechanisms are deployed: end-joining and
homologous recombination (Bishop and
Smith, 1989). In view of the similar structure
of integrated arrays following embryo
microinjection, we have suggested that the
extrachromosomal events have the same
mechanistic basis.
End-joining is the ligation of DNA
duplexes, by blunt-end ligation, or with the
participation of the short complementary
sequences that are exposed upon digestion with a restriction enzyme, or by illegitimate recombination (ie, recombination
between imperfectly or even poorly matched
DNA duplexes). When the ends of the input
DNA molecules are blunt, or have compatible single-strand extensions, we would
expect that neighbouring copies would be
joined with equal frequencies in the same
(direct) and opposite (inverted) orientation.
The evidence from the analysis of integrated
arrays is that directly orientated neighbours
are much more frequent than invertedly orientated neighbours. This indicates that endjoining makes only a minor contribution to
the formation of arrays.
The high frequency of directly repeated
copies points to their origin by homologous
recombination, but this explanation requires
the invocation of a series of events that at
first may seem improbable. Consider a population of identical linear DNA molecules
isolated after restriction enzyme digestion
of a plasmid or bacteriophage. Molecules
within this population can be expected to
participate in end-joining by any of the processes detailed earlier, and none of the evidence is inconsistent with the occurrence
of such a process. We have seen, however,
that this process makes only a minor contribution to the integrated arrays. Homologous recombination between such
molecules would simply generate more identical molecules. How then are we to explain
the extrachromosomal generation of direct
tandem arrays?
We earlier proposed such an intrinsically
improbable series of events. We were able
to show that linear molecules become circularized in transfected cells by the joining
of their ends (Bishop and Smith, 1989): this
is analogous to end-joining of two molecules,
but occurs at a higher frequency under most
circumstances simply because the two ends
a short DNA duplex are always in close
proximity and, therefore, will frequently collide. Another way of looking at this is to
appreciate that the collision of the two ends
of
of the
same
molecule is unimolecular and
independent of DNA concentration. In contrast, the collision of the ends of two DNA
molecules is a bimolecular reaction and
therefore concentration-dependent. Given
a sufficiently high DNA concentration, the
rate of the bimolecular reaction could, in
principle, exceed that of the unimolecular
reaction. We suggest that the effective in
vivo DNA concentration is substantially
below this limit in most if not all cases. Endjoining of input DNA molecules (but without
distinguishing intra- from inter-molecular
associations) has been observed within minutes of microinjecting mouse embryo nuclei
(Burdon and Wall, 1992).
In transfected
cells,
we
also showed
(Bishop and Smith, 1989) that circular
molecules are randomly cleaved by cellular nucleases, generating a population of
DNA molecules best described
as
’circu-
larly permuted linear’ molecules (fig 1iflf
any two of these molecules, linearized by
cleavage in different places, undergo homologous recombination, or if one such
molecule recombines with an input DNA
molecule that has not become circular, then
in each case one of the products will be a linear molecule up to twice as long as the input
linear molecules and, crucially, usually incorporating at least one of the unimolecularly
joined ends (fig 1As a result, the model
accommodates any damage at the junctions between tandem repeats of the input
DNA (Hamada et al, 1993; Chen et al,
1995), and only to a very much lesser extent
’internal’ damage, which would, acccording
to the model, imply multiple rounds of circularization and linearization. Perhaps the
most telling feature of the model is that it
allows for the assembly of perfect tandem
arrays (apart from the junction points) even
from fragments of the circularized molecules
that are less than unit length.
What we
envisaged was that, irrespective
of whether the input DNA molecules were
linear or circular, they would generate concatemers of a size that would in general be
proportional to the square of the DNA input
and with two
important properties, namely
the fidelity of the internal sequences and
the near-identity of what appear to be ’intermolecular junctions’ but which were in fact
intramolecular in the first instance. An important implication of the model is that if overlapping fragments of a larger DNA molecule
introduced into the same cell, the larger
DNA molecule would be reconstituted intracellularly by recombination. Such reconstitutions have been reported in transgenic
animals (Shimoda et al, 1991; Pieper et al,
1992) and in one of the studies (Pieper et al,
1992) a fragmented gene was reconstituted
so faithfully that it was correctly transcribed
and spliced and the mRNA was correctly
translated.
are
What recombinatorial mechanism is
employed is inconsequential to the model
provided only that identical sequences
become recombined. However, a substantial body of work shows that, in transfected
cultured cells, DNA concatemers are built
up by a nonconservative recombination
mechanism (Lin et al, 1984; Wake et al,
1985). As yet there is no reason to suppose
that a different mechanism obtains in
embryos. According to the nonconservative
model, single-stranded 3’ ends are exposed
in linear DNA molecules by the action of a
5’exonuclease (Lin et al, 1987). Collisions
between molecules lead to intermolecular
base-pairing, very likely followed by branch
migration. Recombination is completed by
DNA repair reactions involving endo- or
exonuclease action, ligation and possibly
synthesis (fig 2).
INTEGRATION INTO
THE CHROMOSOME
all of this information is small, we
nevertheless can glean an overall picture
of the processes. Several instances of low
level homology between the transgene and
the chromosomal integration site (Rohan et
al, 1990; Hamada et al, 1993; Allen et al,
1994), and the frequent integration of a minisatellite transgene into a resident satellite
sequence (Allen et al, 1994), both indicate
that ’illegitimate’ recombination between
very poorly matched sequences may be
involved in most if not all cases. There is
considerable diversity among the few wellstudied integrates, suggesting that chance
plays a large part in the outcome. Most often
the chromosome is altered by deletion
provide
(Covarrubias et al, 1987; Gonzalez, 1988;
Xiang et al, 1990; Brown et al, 1994; Naora
et al, 1994; Chen et al, 1995). Other
observed effects are duplication and inversion (Wilkie and Palmiter, 1987), complex
disturbances of the original chromosome at
some distance from the point at which the
foreign DNA is inserted (Covarrubias et al,
1986) and multiple insertions in close proximity to each other (Covarrubias et al, 1986;
Michalova et al, 1988). The most remarkable feature of the junctions between foreign and resident DNA is the inclusion of
copies of sequences from elsewhere in the
genome (Wilkie and Palmiter, 1987) and of
sequences that have no known homology
with either the foreign DNA or the resident
chromosome complement (Wilkie and
Palmiter, 1987; Chen et al, 1995). Since we
not aware of such occurrences in the
normal course of cell proliferation, we must
surmise that they are provoked by the presence of the foreign DNA. It is tempting to
suppose that single-stranded ends of extrachromosomal DNA molecules, exposed by
the 5’exonuclease referred to earlier, invade
DNA duplexes to initiate the process of integration. This would be sufficient to explain
deletions of resident DNA, by the invasion of
a DNA duplex by two exposed ends of an
extrachromosomal molecule at two different points, followed by replacement of the
are
Following the proposed extrachromosomal
concatenation of foreign DNA, one or more
molecules, generally catenated, are linearly
inserted into the DNA duplex of one or rarely
chromosome. We can expect
gain
understanding of the processes involved from an analysis of the
nucleotide sequences at the junctions
between foreign and resident DNA, and of
the pre-insertion sequences of the resident
chromosomal site and the input foreign
DNA. Although the number of studies that
more
to
than
one
some
intervening resident
DNA
by the foreign
DNA.
To provide an explanation for one particular well-studied integration event, Wilkie
and Palmiter (1987) proposed that a replication ’eye’ had been invaded by the extrachromosomal DNA. This provides an attractive general model for DNA insertion for
several reasons. Most importantly, we can
imagine that the replication eye is more
accessible to invasion than a nonreplicating DNA duplex. In addition, regions where
replication is underway can be supposed to
be populated by enzymes that carry out
DNA synthesis and repair. Replication eyes
also offer an explanation of how invading
DNA might initiate duplications of resident
DNA (Wilkie and Palmiter, 1987; Bishop and
Smith, 1989). It has recently become apparent that the majority of transgenic founders
are mosaics of transgenic and non-transgenic cells, despite the DNA having been
injected at the one-cell stage (Whitelaw et al,
1993; Ellison et al, 1995). This does not
prove that integration occurs during DNA
replication, but it is certainly consistent with
that idea. It is quite possible that the minority (ca 15%) of nonmosaic founders arise
in the same way but with the death of the
nontransgenic daughter cell due to chomosomal damage. If so, some of the nontransgenic siblings of transgenic founders
may contain nonlethal chromosomal rearrangements, the transgenic daughter cell
having been lost following DNA integration.
These possibilities are supported by the
negative effect of microinjected DNA on
embryo viability (Page et al, 1995).
ABERRANT EXPRESSION
OFTRANSGENES
There is a tendency for the expression of
DNA sequences introduced into a foreign
genome by random integration to suffer a
number of aberrations, namely poor (low
level) expression, temporally (developmental) or spatially (ectopic) aberrant
expression and expression that is related
to chromosomal position (type I). These
aberrations are accompanied by a lack of
correlation between the number of copies
of the gene in the integrated array and the
level of expression, which is called copynumber independence. This could be taken
to indicate that the effects of chromosomal
position on expression overwhelm the
effects of copy-number. A smaller but growing number of transgenes do not show these
effects and instead show a high level and
often developmentally correct expression
which is also copy-number dependent (type
II). The difference between the two groups
of genes is essentially trivial, in a scientific
sense, in that the weight of evidence suggests that, given the inclusion in a transgene of sufficient flanking sequence, it will
revert to type I expression. Type I expression will often reflect the lack of an enhancer
sequence. The best studied examples of
remote sequences required for high level
and correct expression, the a- and (3-globin
locus control regions (LCRs) are effectively
tissue-specific enhancer sequences
(Grosveld et al, 1987; Caterina et al, 1991;
Pondel et al, 1992).
When we move away from simply introducing a foreign gene to directing the
expression of a foreign protein in a tissue
in which it is not normally produced, we raise
a number of new problems. An example
would be the production of transgenic sheep
to secrete a-1-antitrypsin in their milk. Here
the expression of a coding sequence derived
from a human gene is driven by a sheep
gland-specific promoter
(Archibald et al, 1990). What if, as is frequently the case, the activity of the promoter
depends on an enhancer sequence contained within an intron, and especially if the
mammary
enhancer is downstream of the initiation
codon for translation? Questions arising
from this scenario are addressed here.
THE WAY FORWARD
occupy
a
separate chromosomal ’domain’
et al, 1989). Although this approach
has not been much exploited, encouraging
results have been reported (McKnight et al,
(Stief
There
are
potentially at least three general
problems of expression,
ways to deal with
which we will call
head-on, circumvention
and avoidance.
1992). The activation of a relatively inert
by admixture with an active gene is
perhaps a similar approach (Clark et al,
1992).
gene
Head-on
Avoidance
It is evident that the more that we know
about the expression requirements of a
given promoter the better position we are in
to utilise it effectively. Essential enhancer
sequences, whether upstream, downstream
or intronic, will usually be detectable by
their DNase-hypersensitivity (Caterina et
al, 1991; Lowrey et al, 1992). There is evidence that remote enhancer sequences will
work perfectly well when brought into closer
proximity with the promoter (Grosveld et al,
1987; Bonnerot and Nicolas, 1993). Intronic
enhancer sequences, which in the majority
of cases are located in the first intron, pose
a greater problem. It is not known whether
they can be successfully relocated. If not,
and if the first exon includes the translational initiation codon, then this could be
removed. Given that the longer leader
sequence thus generated does not include
a stable secondary structure, translation
should not be impaired (Kozak, 1991,
1994). A stable secondary structure could of
course be removed. It is possible, although
by no means certain, that the head-on
approach might eliminate position effect in
all cases.
Circumvention
There is a class of sequence, the matrix
attachment sequences (MAR) which, when
placed on either side of a gene, is believed
to insulate it from chromosomal position
effect, possibly by allowing the gene to
If ES cells could be obtained from all types
of mammals, the problems of expression
could be solved readily. Transgenes would
be engineered with the appropriate flanking sequences and substituted for high
expression genes. In most cases the
remaining haploid normal gene would be
able to cope with the demand for its normal
function. However, to date ES cells can only
be obtained from mice, and then only from
particular strains of mice (Smith, 1992). The
correlation that exists with ease of production of embryonal carcinomas by ectopic
implantation suggests that the inbred
(homozygous) strains of mice in question
may carry mutations that are essential to
the generation of ES cells. We cannot doubt
that ES cells will be obtained from other
mammals in time, but the time-scale may
be very long and it is quite possible that the
donors will have to be engineered genetically so as to make differentiation lead to
death under special in vitro conditions.
If totipotent ES cells cannot be derived
from other species, or until they can, it may
be possible to rescue embryonic cell lines
that have lost the capacity to contribute to a
chimera by transplanting their nuclei into
enucleated or parthenogenetically activated
one-cell embryos. Some success in this
direction has been reported (Campbell et
al, 1996). If this can be done successfully
it could be a short step to obtaining gene
substitution by homologous recombination
via this route.
Another approach would be to harness
site-specific recombination. There are two
superfamilies of site-specific recombinases,
integrases and resolvases, which have different reaction mechanisms. Recent attention has focused on the integrases, and in
particular the CRE-/oxP system of bacteriophage P1 and the FLP-FRS yeast system, which are the simplest of the superfamily, neither requiring the participation of
other proteins in the recombination event.
Their essential property is that they effect
recombination with a very high degree of
specificity between two identical sites (lox
p
or FRS!. These sites contain inverted repeat
sequences that bind the enzyme (CRE or
FLP) surrounding a short unique sequence
that confers orientation on the site. Thus,
CRE-mediated recombination between two
p sites on the same
directly-orientated lox
molecule results in a deletion of the intervening DNA, while recombination between
p sites results in
oppositely-orientated lox
an inversion of the intervening DNA (Kilby et
al, 1993) (see fig 3). These systems are
capable of operating in mammalian cells,
and have been employed to effect tissuespecific deletions of DNA (Lakso et al, 1992;
Sauer, 1993). As opposed to the highly efficient deletion reaction, their capacity to promote the integration of a DNA molecule into
a chromosomal site is minimal (Fukushige
and Sauer, 1992). This may possibly be due
simply to a relatively much higher frequency
of the excision reaction. This would be due
p sites that parto the proximity of the lox
ticipate in excision, located at no great distance from each other on the same DNA
molecule. In contrast, integration would follow the collision of a foreign DNA molecule
with a single chromosomal site within the
entire genome. Thus, the balance of the
reactions mediated by CRE recombinase
may strongly favour deletion. If this problem can be overcome it may be possible to
introduce foreign DNA sequences into
selected high expression sites by site-specific recombination.
REFERENCES
Allen MJ, Jeffreys AJ, Surani MA, Barton S, Norris ML,
Collick A (1994) Tandemly repeated transgenes of
the human minisatellite MS32 (D1S8), with novel
mouse gamma satellite integration. Nucleic Acids
Res 22, 2976-2981
Archibald AL, McCienaghan M, Hornsey V, Simons JP,
Clark AJ (1990) High-level expression of biologically
active human alpha 1-antitrypsin in the milk of transgenic mice. Proc Natl Acad Sci USA 87, 5178-5182
Bishop JO, Smith P (1989) Mechanism of chromosome
integration of microinjected DNA. Mol Biol Med 6,
283-298
Campbell KH, McWhir J, Ritchie WA, WilmutI (1996)
Sheep cloned by nuclear transfer from a cultured
cell line [see comments]. Nature 380, 64-66
Caterina JJ, Ryan TM, Pawlik KM, Palmiter RD, Brinster RL, Behringer RR, Townes TM (1991) Human (3globin locus control region: analysis of the 5’-DNase
I hypersensitive site HS 2 in transgenic mice. Proc
Natl Acad Sci USA 88, 1626-1630
Chen CM, Choo KB, Cheng WT (1995) Frequent deletions and sequence aberrations at the transgene
junctions of transgenic mice carrying the papillomavirus regulatory and the SV40 TAg gene
sequences. Transgenic Res 4, 52-59
Cowper A, Wallace R, Wright G, Simons JP
(1992) Rescuing transgene expression by co-integration. Biotechnologyl0, 1450-1454
Covarrubias L, Nishida Y, Mintz B (1986) Early postimplantation embryo lethality due to DNA rearrangements in a transgenic mouse strain. Proc Natl Acad
Clark AJ,
Sci USA 83, 6020-6024
Covarrubias L, Nishida Y, Terao M, D’Eustachio P, Mintz
B (1987) Cellular DNA rearrangements and early
developmental arrest caused by DNA insertion in
transgenic mouse embryos. Mol Cell Biol7, 22432247
Cramer CL, Weissenborn DL, Oishi KK, Grabau EA,
Bennett S, Ponce E, Grabowski GA, Radin DN
(1996) Bioproduction of human enzymes in transgenic tobacco [review]. Ann NYAcad Sci 792, 62-71
Ellison AR, Wallace H, Al-Shawi R, Bishop JO (1995)
Different transmission rates of herpes virus thymidine kinase reporter transgenes from founder male
parents and male parents of subsequent generations. Mol Reprod Dev 41, 425-434
Fukushige S, Sauer B (1992) Genomic targeting with a
positive-selection lox integration vector allows highly
reproducible gene expression in mammalian cells.
Proc Natl Acad Sci USA 89, 7905-7909
Gonzalez FJ (1988) The molecular biology of cytochrome
P450s [published erratum appears in Pharmacol
Rev 1989; 41:91-92] [review]. Pharmacol Rev 40,
243-288
Grosveld F,
van
Assendelft GB, Greaves DR, Kollias G
(1987) Position-independent, high-level expression
of the human beta-globin gene in transgenic mice.
Cell51, 975-985
(1993) Lineage control of an
integration site-dependent transgene combined with
the beta-globin locus control region. C R Acad Sci
Hackland AF, Rybicki EP, Thomson JA (1994) Coat protein-mediated resistance in transgenic plants [review].
Arch Virol139, 1-22
Serie lii, Sciences de la Vie 316, 352-357
Hamada T, Sasaki H, Seki R, Sakaki Y (1993) Mechanism of chromosomal integration of transgenes in
microinjected mouse eggs: sequence analysis of
genome-transgene and transgene-transgene junctions at two loci. Gene 128, 197-202
Bonnerot C, Nicolas JF
Copeland NG, Gilbert DJ, Jenkins NA, Rossant
J, Kothary R (1994) The genomic structure of an
insertional mutation in the dystonia musculorum
Brown A,
locus. Genomics 20, 371-376
Burdon TG, Wall RJ (1992) Fate of microinjected genes
in preimplantation mouse embryos. Mol Reprod Dev
33, 436-442
Hoyer LW, Drohan WN, Lubon H (1994) Production of
human therapeutic proteins in transgenic animals
[review]. Vox Sanguinis 67 (suppl 3), 217-220
Janne J, Hyttinen JM, Peura T, Tolvanen M, Alhonen
L, Sinervirta R, Halmekyto M (1994) Transgenic
bioreactors [review]. Int J Biochem 26, 859-870
Kilby NJ, Snaith MR, Murray
JA (1993) Site-specific
recombinases: tools for genome engineering. Trends
Genet 9, 413-4211
Kozak M (1991) Effects of long 5’ leader sequences on
initiation by eukaryotic ribosomes in vitro. Gene Expr
1, 117-125
Kozak M (1994) Features in the 5’ non-coding sequences
of rabbit alpha and beta-globin mRNAs that affect
translational efficiency. J Mol Bio1235, 95-110
0
Lakso M, Sauer B, Mosinger B, Lee EJ, Manning RW, Yu
SH, Mulder KL, Westphal H (1992) Targeted oncogene activation by site-specific recombination in
transgenic mice. Proc Natl Acad Sci USA 89, 62326236
Lin FL, Sperle K, Sternberg N (1984) Model for homologous recombination during transfer of DNA into
mouse L cells: role for DNA ends in the recombination process. Mol Cell BioI 4, 1020-1034
Pieper FR, de Wit IC, Pronk AC, Kooiman PM, Strijker R,
Krimpenfort PJ, Nuyens JH, de Boer HA (1992) Efficient generation of functional transgenes by homologous recombination in murine zygotes. Nucleic
Acids Res 20, 1259-1264
Pondel MD, Proudfoot NJ, Whitelaw C, Whitelaw E
(1992) The developmental regulation of the human
zeta-globin gene in transgenic mice employing betagalactosidase as a reporter gene. Nucleic Acids Res
20, 5655-5660
Robertson EJ (1991) Using embryonic stem cells to
introduce mutations into the mouse germ line. Biot
Reprod 44, 238-245
Rohan RM, King D, Frels Wi (1990) Direct sequencing
of PCR-amplified junction fragments from tandemly
repeated transgenes. Nucleic Acids Res 18, 60896095
Sauer B (1993) Manipulation of transgenes by site-specific recombination: use of Cre recombinase. Methods Enzymol 225, 890-900
Shimoda K, Cai X, Kuhara T, Maejima K (1991) Reconstruction of a large DNA fragment from coinjected
small fragments by homologous recombination in
fertilized mouse eggs. Nucleic Acids Res 19, 6654
Lin FL, Sperle KM, Sternberg NL (1987) Extrachromosomal recombination in mammalian cells as studied with single- and double-stranded DNA substrates.
Mol Cell Biol7, 129-140
Smith AG
Lowrey CH, Bodine DM, Nienhuis AW (1992) Mechanism of DNase I hypersensitive site formation within
the human globin locus control region. Proc Natl
tification, propagation and manipulation. Sem Cell
Biol3, 385-399
Stief A, Winter DM, Stratling WH, Sippel AE (1989) A
Acad Sci USA 89, 1143-1147
McKnight RA, Shamay A, Sankaran L, Wall RJ, Hennighausen L (1992) Matrix-attachment regions can
impart position-independent regulation of a tissuespecific gene in transgenic mice. Proc Natl Acad Sci
USA 89, 6943-6947
Michalova K, Bucchini D, Ripoche MA, Pictet R, Jami J
(1988) Chromosome localization of the human insulin
gene in transgenic mouse lines. Hum Genet 80, 247252
Naora H, Kimura M, Otani H, Yokoyama M, Koizumi T,
Katsuki M, Tanaka 0 (1994) Transgenic mouse
model of hemifacial microsomia: cloning and characterization of insertional mutation region on chromosome 10. Genomics 23, 515-519
9
Page RL, Canseco RS,
Russell CG, Johnson JL,
Velander WH, Gwazdauskas FC (1995) Transgene
detection during early murine embryonic development after pronuclear microinjection. Transgenic
Res 4, 12-17
7
(1992)
Mouse
embryo stem
cells: their iden-
nuclear DNA attachment element mediates elevated
and position-independent gene activity. Nature 341,
343-345
Tabe LM, Higgins CM, McNabb WC, Higgins TJ (1993)
Genetic engineering of grain and pasture legumes for
improved nutritive value [review]. Genetica 90, 181200
Wake
CT, Vernaleone F, Wilson JH (1985) Topological
requirements for homologous recombination among
DNA molecules transfected into mammalian cells.
Mol Cell BiolS, 2080-2089
Whitelaw CB, Springbett AJ, Webster J, Clark J (1993)
The majority of GO transgenic mice are derived from
mosaic embryos. Transgenic Res 2, 29-32
Wilkie TM, Palmiter RD (1987) Analysis of the integrant
in MyK-103 transgenic mice in which males fail to
transmit the integrant. Mol Cell Biol7, 1646-1655
Xiang X, Benson KF, Chada K (1990) Mini-mouse: disruption of the pygmy locus in a transgenic insertional
mutant. Science
247, 967-9 69