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Claims  |
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We claim:
1. A process for the high efficiency rescue and cloning of a DNA fragment
from genomic mammalian DNA comprising the steps of preparing a vector
containing the DNA fragment, followed by multiplying said vector
containing said DNA fragment in a suitable bacterial host wherein said
bacterial host is selected as being incapable of host restriction and
further is selected as being capable of rescuing 900,000 copies of said
fragment per 100 micrograms of genomic mammalian DNA when said 100
micrograms of genomic mammalian DNA derives from a mouse genome in which
said fragment has a copy number of about 80 and said about 80 copies are
present in a head-to-tail arrangement to effect rescue and cloning of said
DNA fragment at an efficiency level defined as that efficiency of which a
bacterial host, selected as being capable of rescuing 900,000 copies of
said fragment per 100 micrograms of genomic mammalian DNA when said 100
micrograms of genomic mammalian DNA derives from a mouse genome in which
said fragment has a copy number of about 80 and said about 80 copies are
present in a head-to-tail arrangement, is capable.
2. The process according to claim 1 wherein said bacterial host is an
Escherichia coli strain.
3. The process according to claim 1 wherein said bacterial host is an
Escherichia coli C strain.
4. A process according to claim 1 wherein said vector is a bacteriophage
vector obtained by means of a phage packaging extract derived from a
bacterial host selected as being incapable of host restriction and further
selected as being capable of rescuing 900,000 copies of said fragment per
100 micrograms of genomic mammalian DNA when said 100 micrograms of
genomic mammalian DNA derives from a mouse genome in which said fragment
has a copy number of about 80, said about 80 copies are present in a
head-to-tail arrangement.
5. A process according to claim 1 wherein said vector is a plasmid.
6. A process for the rescue and cloning of a DNA fragment from genomic
mammalian DNA wherein a vector containing said DNA fragment is multiplied
in a first bacterial host, wherein said first bacterial host is selected
as being incapable of host restriction and further is capable of rescuing
900,000 copies of said fragment per 100 micrograms of genomic mammalian
DNA when said 100 micrograms of genomic mammalian DNA derives from a mouse
genome in which said fragment has a copy number of about 80, said about 80
copies are present in a head-to-tail arrangement, further wherein said
first bacterial host is an Escherichia coli strain, further wherein said
vector is a bacteriophage lambda vector, and further wherein said
bacteriophage lambda vector is obtained by means of a phage packaging
extract derived from a second bacterial host incapable selected as being
of host restriction and further selected as being capable of rescuing
900,000 copies of said fragment per 100 micrograms of genomic mammalian
DNA when said 100 micrograms of genomic mammalian DNA derives from a mouse
genome in which said fragment has a copy number of about 80, said about 80
copies are present in a head-to-tail arrangement, to effect rescue and
cloning of the genomic mammalian DNA fragment.
7. The process according to claim 6 wherein each of said first bacterial
host and said second bacterial host is an Escherichia coli C strain.
8. A process for detecting mutations in one or more marker genes introduced
into a mammalian genome by means of a vector, which process comprises
isolating DNA from cells of a transgenic mammal or from mammalian cells,
recovering said DNA by means of a vector, multiplying said vector in a
bacterial host which is deficient with respect to at least one of the
marker genes, and testing marker gene expression for mutation, wherein
said bacterial host is selected as being incapable of host restriction and
further is capable of rescuing 900,000 copies of a fragment containing
said vector per 100 micrograms of genomic mammalian DNA when said 100
micrograms of genomic mammalian DNA derives from a mouse genome in which
said fragment has a copy number of about 80 and said about 80 copies are
present in a head-to-tail arrangement, wherein said recovering and
multiplying of said DNA is performed at an efficiency level defined as
that efficiency of which a bacterial host, selected as being capable of
rescuing 900,000 copies of said fragment per 100 micrograms of genomic
mammalian DNA when said 100 micrograms of genomic mammalian DNA derives
from a mouse genome in which said fragment has a copy number of about 80
and said about 80 copies are present in a head-to-tail arrangement, is
capable.
9. The process according to claim 8 wherein said bacterial host is an
Escherichia coli strain.
10. The process according to claim 8 wherein said bacterial host is an
Escherichia coli C strain.
11. The process according to claim 8 wherein said vector is a bacteriophage
vector obtained by means of a phage packaging extract derived from a
bacterial host selected as being incapable of host restriction and further
selected as being capable of rescuing 900,000 copies of said fragment per
100 micrograms of genomic mammalian DNA when said 100 micrograms of
genomic mammalian DNA derives from a mouse genome in which said fragment
has a copy number of about 80, and said about 80 copies are present in a
head-to-tail arrangement.
12. The process according to claim 8 wherein said vector is a plasmid.
13. A process for detecting mutations in one or more marker genes
introduced into a mammalian genome by means of a vector, which process
comprises isolating DNA from cells of a transgenic mammal or from
mammalian cells, recovering said DNA by means of a vector, multiplying
said vector in a first bacterial host which is deficient with respect to
at least one of the marker genes, and testing marker gene expression for
mutation, wherein said first bacterial host is an Escherichia coli strain
selected as being incapable of host restriction and further selected as
being capable of rescuing 900,000 copies of a fragment containing said
vector per 100 micrograms of genomic mammalian DNA when said 100
micrograms of genomic mammalian DNA derives from a mouse genome in which
said fragment has a copy number of about 80, said about 80 copies are
present in a head-to-tail arrangement, and further wherein said vector is
a bacteriophage vector obtained by means of a phage packaging extract
derived from a second bacterial host selected as being incapable of host
restriction and further selected as being capable of rescuing 900,000
copies of said fragment per 100 micrograms of genomic mammalian DNA when
said 100 micrograms of genomic mammalian DNA derives from a mouse genome
in which said fragment has a copy number of about 80 and said about 80
copies are present in a head-to-tail arrangement.
14. The process according to claim 6 wherein each of said first bacterial
host and said second bacterial host is an Escherichia coli C strain.
15. The process according to any of claims 8-14 characterized in that the
DNA isolated from cells of the transgenic mammal or the mammalian cells is
prepurified by fragmenting the DNA by means of a restriction enzyme which
does not have a cutting site within the vector, separating the resulting
fragments on the basis of a suitable criterion, such as differences in
size, and collecting fragments comprising the vector, whereafter the
vector is recovered from said prepurified DNA.
16. A process for detecting mutations in one or more marker genes
introduced into a mammalian genome by means of a vector, which process
comprises isolating DNA from cells of a transgenic mammal or from
mammalian cells, recovering said DNA by means of a vector, multiplying
said vector in a first bacterial host which is deficient with respect to
at least one of the marker genes, and testing marker gene expression for
mutation, wherein said first bacterial host is an Escherichia coli strain
selected as being incapable of host restriction and further selected as
being capable of rescuing 900,000 copies of a fragment containing said
vector per 100 micrograms of genomic mammalian DNA when said 100
micrograms of genomic mammalian DNA derives from a mouse genome in which
said fragment has a copy number of about 80, said about 80 copies are
present in a head-to-tail arrangement, and further wherein said vector is
a bacteriophage lambda vector obtained by means of a phage packaging
extract derived from a second Escherichia coli bacterial host incapable
selected as being of host restriction and further selected as being
capable of rescuing 900,000 copies of said fragment per 100 micrograms of
genomic mammalian DNA when said 100 micrograms of genomic mammalian DNA
derives from a mouse genome in which said fragment has a copy number of
about 80, said about 80 copies are present in a head-to-tail arrangement
further wherein said bacteriophage lambda vector has a size of at least 40
kb, further wherein at least 3 copies of said vector are integrated in the
genome of said transgenic mammal or said mammalian cells in a head to tail
arrangement and further wherein said DNA is prepurified by fragmenting the
DNA by means of the restriction enzyme XbaI, which does not have a cutting
site within the vector but does have such a large number of cutting sites
in the mammalian genome that it is capable of generating fragments having
an average size below 10 kb, separating the resulting fragments on the
basis of their size differences, and collecting the larger fragments
comprising said vector.
17. The process according to claim 16 wherein each Escherichia coli strain
is an Escherichia coli C strain.
18. The process according to claim 16 wherein at least 10 copies of said
vector are integrated in the genome of said transgenic mammal or said
mammalian cells in a head to tail arrangement.
19. A process for detecting mutations in one or more marker genes
introduced into a mammalian genome by means of a vector, which process
comprises isolating DNA from cells of a transgenic mammal or from
mammalian cells, recovering the vector from said DNA, multiplying said
vector in an Escherichia coli bacterial host incapable of host selected as
being restriction and further selected as being capable of rescuing
900,000 copies of a fragment containing said vector per 100 micrograms of
genomic mammalian DNA when said 100 micrograms of genomic mammalian DNA
derives from a mouse genome in which said fragment has a copy number of
about 80, said about 80 copies are present in a head-to-tail arrangement
and testing marker gene expression for mutation, wherein said vector is
further a plasmid vector flanked by restriction enzyme cutting sites which
do not occur within the vector, and said vector is recovered by
fragmenting the DNA isolated from cells of the transgenic mammal or the
mammalian cells by means of a restriction enzyme specific for said
restriction enzyme cutting sites and by ring closure, and wherein said
recovering and multiplying of said vector is performed at an efficiency
level defined as that efficiency of which a bacterial host, selected as
being capable of rescuing 900,000 copies of said fragment per 100
micrograms of genomic mammalian DNA when said 100 micrograms of genomic
mammalian DNA derives from a mouse genome in which said fragment has a
copy number of about 80 and said about 80 copies are present in a
head-to-tail arrangement, is capable.
20. A process for detecting mutations in one or more marker genes
introduced into a mammalian genome by means of a vector, which process
comprises isolating DNA from cells of a transgenie mammal or from
mammalian cells, recovering said DNA by means of a vector, multiplying
said vector in a first bacterial host which is deficient with respect to
at least one of the marker genes, and testing marker gene expression for
mutation, wherein said first bacterial host is an Escherichia coli strain
selected as being incapable of host restriction and further selected as
being capable of rescuing 900,000 copies of a fragment containing said
vector per 100 micrograms of genomic mammalian DNA when said 100
micrograms of genomic mammalian DNA derives from a mouse genome in which
said fragment has a copy number of about 80, said 80 copies are present in
a head-to-tail arrangement, and further wherein said vector is a plasmid
vector having a size of not more than 10 kb flanked by NotI restriction
enzyme cutting sites which do not occur within said vector and which occur
in such a small number in the genome of said transgenic mammal or
mammalian cells that they are capable of generating DNA fragments having
an average size greater than 20 kb, and further wherein the DNA isolated
from the cells of said transgenic mammal or said mammalian cells is
prepurified by fragmenting said isolated DNA with a restriction enzyme
specific for said NotI restriction sites, separating the resulting
fragments on the basis of their size differences and collecting the small
fragments comprising said vector and wherein said recovering and
multiplying of said DNA is performed at an efficiency level defined as
that efficiency of which a bacterial host, selected as being capable of
rescuing 900,000 copies of said fragment per 100 micrograms of genomic
mammalian DNA when said 100 micrograms of genomic mammalian DNA derives
from a mouse genome in which said fragment has a copy number of about 80
and said about 80 copies are present in a head-to-tail arrangement, is
capable. |
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Claims |
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Description |
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This invention relates to a process for the efficient rescue and cloning of
DNA sequences from genomes in general, in particular methylated DNA
sequences, and relates more specifically to a process for detecting
mutations in one or more marker genes introduced into a mammalian genome
by means of a vector, which process comprises isolating DNA from cells of
the transgenic mammal or from the mammalian cells, recovering the vector
from said DNA, multiplying the vector in a suitable bacterial host
deficient with respect to at least one of the marker genes, and testing
the expression of the marker gene.
Such a process is known from the articles by Lohman et al. 1987; and Vijg
and Uitterlinden, 1987, disclosing an animal test model, inter alia for
estimating the risks of potentially carcinogenic agents. More in
particular, they propose a mouse model for in vivo mutation analysis using
transgenic mice of which all cells, including the germ cells, comprise a
bacteriophage lambda vector integrated in the genome, which vector is
provided with a selectable marker. Testing of agents for carcinogenicity
is of great importance in a society in which more and more newly
synthesized agents make their appearance. Many of these agents, as well as
a number of already existing chemical compounds are suspected of
carcinogenicity. According to present day insights cancer would be based
on changes in the DNA base sequence of certain genes. Mutagenic agents
(i.e. agents that change the base sequence of the DNA) are nearly always
carcinogenic and can also cause permanent damage to the heritable material
(and therefore lead to hereditary diseases). Consequently, the ability of
an agent to introduce mutations into genes is considered to be an
important criterion for estimating the risks of the relevant agent as
regards injurious effects on health, such as cancer and deviations in the
hereditary information.
The best known test for potentially mutagenic agents is the Ames test
dealing with mutations in bacteria (Ames et al., 1973). Probably, because
bacteria are rather remote from man, this test is not 100% reliable
(Zeiger et al., 1987). For this reason preference is often given to a
combination of tests, including tests with mouse cells. As applies to all
mammalian cells, the genome of mouse cells is too complicated for mutation
studies analogous to the Ames test; a great advantage of bacteria is that
they have very short generation times, which enable selection for certain
mutations, as well as their characterization.
An important problem with cultured mammalian cells as a model in
mutagenicity tests is the artificial aspect of the in vitro situation and
the impossibility to test for differences between organs and tissues as
regards nature and frequency of induced mutations. For instance, it may be
very important to know whether a certain agent preferably induces
mutations in the germ cells, or just the reverse. On the other hand,
information is necessary with respect to the possibility of drawing
conclusions on mutations in certain organs on the basis of measurements on
blood cells. This is important, for instance, when screening people who
may have been exposed to dangerously high doses of a carcinogenic agent.
At this moment there are only few possibilities of testing mutations in
vivo in cells of higher animals. An example is the so-called HPRT test in
which mutations in the HPRT gene are selected on the basis of the
6-thioguanine resistance of the isolated and cultured cells (Albertini et
al., 1982). This method is labour-intensive and only allows measurements
on cells that can still divide and are easy to culture. In addition the
HPRT method is prone to artefacts (Featherstone et al., 1987).
The approach suggested in the above-mentioned articles by Lohman et al.,
1987 and Vijg and Uitterlinden, 1987, implies that a bacteriophage lambda
vector integrated in the mouse DNA, which vector contains one or more
selectable bacterial genes, can be recovered from total chromosomal DNA by
mixing this DNA with an enzyme cocktail which cuts the vector at the
flanking "cos sites" from the mouse DNA and then encapsulates it.
Subsequently, the packaged phages together with an excess of bacteria are
plated on a nutrient medium, which after a few hours results in a number
of so-called plaques, i.e. circular clear areas in the bacterial lawn.
Each plaque corresponds to one recovered vector. If the marker gene in the
phage is intact, it will be expressed. The absence of expression is
therefore indicative of a mutation in the marker gene.
For completeness' sake, it should be mentioned here that the above approach
cannot be adopted with naturally present genes. These simply cannot be
isolated from the DNA of an organ with great efficiency, in contrast to
the integrated vector, which has special properties including the presence
of two half cos sites at both sides of the vector. If the vector is
integrated in the mouse DNA in a head-to-tail arrangement, the central
vectors can be cut from the mouse DNA at the whole cos sites. It is
therefore essential, at least with vectors flanked by half cos sites, that
at least 3 vectors integrate in a head-to-tail arrangement. In the case of
vectors flanked by whole cos sites this is not necessary.
An example of a marker gene is the bacterial gene lacZ. With this (and with
other corresponding marker genes) frequency and nature of mutations in
cells or in the different organs and tissues of a transgenic animal can be
simply determined by means of the above described recovery of the vector
and its transfer to bacteria. The lacZ gene codes for .beta.-galactosidase
which converts X-gal (the substrate) to a blue substance. X-gal can be
added to the nutrient medium on which the bacteriophages are plated with
an excess of bacteria. Each phage will give rise to a blue plaque if the
lacZ gene is still intact. Mutations in this gene reveal themselves by
leaving the plaques uncoloured as a result of the inactivation of the lacZ
gene. Each uncoloured plaque is a small reservoir of mutated genes which
can be easily analyzed further if this is deemed necessary. There are a
number of different mutation target genes (lacZ, lacI, lacZ.alpha., supF,
cI, galK, LacO, gpt, etc.), a number of which can be incorporated into a
vector simultaneously or separately.
The above described system can only operate if the vector can be recovered
with a sufficiently high degree of efficiency. In theory, one would like
to "rescue" a vector from each cell of a certain organ in order to
establish whether a mutation has occurred therein. In practice, one can be
satisfied with about 100,000 vectors from a certain organ. For on the
basis of the above described HPRT tests (in white blood cells) the
assumption is that the spontaneous mutation frequency is at most about 1
to 100,000 (Albertini et al., 1985), i.e. out of 100,000 cells in normal
adult individuals, on the average about one or less will be inactivated
for each gene by a mutation at a certain site. This applies to
protein-coding, naturally present genes.
Up to now, there are no indications that bacteriophage lambda vectors (or
other integrated vectors) can be recovered in more than a few tens per
.mu.g genomic DNA (Lindenmaier et al., 1982; Glazer et al., 1986). A very
large amount of phage packaging extract (the enzyme cocktail) is necessary
to rescue the integrated vectors from more than a few micrograms of
genomic DNA. In view of the high price of the present commercially sold
phage packaging extracts a simple scale-up of the procedure for the
purpose of recovering more vectors is not practical. The low phage
packaging efficiency may be the reason that up to now the above described
system has not been used as a mutation model, neither in the form of cell
lines nor in the form of transgenic mice. It is true that extensive use is
made of vectors having mutation target genes thereon, but always without
allowing them to integrate (see for a survey Lehmann, 1985 and Dubridge
and Calos, 1988). An exception is the work by Glazer et al. (1986),
according to which a bacteriophage lambda vector provided with a bacterial
mutation target gene was brought into a cell line by means of
transfection. The integrated gene was then recovered and analyzed for
mutations. The efficiency of the recovery, however, was low.
Recently, the present inventor and colleagues have succeeded in making
transgenic mice with in each cell one or more copies of a bacteriophage
lambda vector provided with a lacZ mutation target gene. In the animals
with at least 3 or more tandem integrated vectors, in vitro packaging into
phage particles from total genomic mouse DNA is possible in principle. In
the first instance, however, it turned out again that the vector could not
be recovered with a fair degree of efficiency.
The present invention provides a solution to this problem and thereby
provides a method for the efficient rescue and cloning of methylated DNA
in general, and more specifically for the efficient rescue of integrated
vectors from total chromosomal DNA of different organs and tissues of a
transgenic mammal or from total chromosomal DNA of a cultured mammalian
cell line (in particular, this is to be taken to mean a cell line obtained
by transfection with the vector, or more in particular, any mammalian cell
line containing the vector, irrespective of how the cell line has been
obtained).
The invention is based on the insight resulting from further research that
the integrated vectors were completely methylated with all tested mice. In
other words, in the organs and tissues of the tested transgenic mouse
strains all tested cytosine residues in the vector were found to be
provided with a covalently bound 5-methyl group. It is known that certain
forms of methylation render bacteria capable of so-called host restriction
(Raleigh et al., 1986). That is to say, entering DNAs (such as, for
instance, the bacteriophage lambda vectors) are cut into pieces by way of
defence before they get the chance of controlling and finally killing
their host (which gives rise to lytic plaques). It was conceived that this
phenomenon might be accountable for the low number of plaques obtained in
practice. Because the conventional, commercially sold phage packaging
extracts are made from bacteria and bacteria are also necessary to plate
the phages (as a host), it was decided for both purposes to work only with
bacterial strains which are incapable of host restriction. To this end,
the phage packaging extracts could be obtained from Stratagene (Stratagene
Cloning Systems, 11099 North Torrey Pines Road, LaJolla, Calif. 92037
USA), which in contrast to other firms supplies phage packaging extracts
that according to our experiments probably originate from E. coli C, a
strain incapable of host restriction. The latter has never been explicitly
mentioned by this firm in their brochures or in their manuals. For
plating, however, the firm does not supply a host restriction-negative
strain, such as E. coli C, together with the extracts, but other strains
which happen to be capable of host restriction. The necessity to use a
packaging extract derived from a host restriction-negative E. coli strain
in combination with the same, or a comparable, host restriction-negative
strain for plating, has never been reported to be necessary for the rescue
of methylated DNA. This is illustrated by the present lack of cloning
systems for methylated mammalian DNA. The E. coli C strain, used in the
studies that gave rise to this invention, is known per se and, for
instance, obtainable from the Phabagen collection (University of Utrecht,
Department of Molecular Cell Biology, Padualaan 8, P.O. Box 80.056, 3508
TB Utrecht). By means of gamma radiation variants were then obtained which
lacked the entire lacZ gene. This was necessary for the purposes of the
experiments described herein, because otherwise selection for inactivation
of lacZ in the vectors to be packaged was not possible.
Using the obtained E. coli C lacZ.sup.- strain, about 9,000 vectors were
recovered from about 1 .mu.g total genomic DNA of one transgenic mouse
strain. The blue colour of the plaques suggested that in all these cases
the lacZ gene was not mutated.
This phage packaging efficiency is by far the highest ever obtained from
total chromosomal DNA and is in principle sufficient for research into
induced mutations.
While the essence of the invention resides in the use of bacterial strains
incapable of host restriction, such as preferably E. coli C strains, it
has further been found conducive to a high phage packaging frequency that
a large number of copies of the vector is integrated in the mammalian
genome in a head-to-tail arrangement. In the transgenic mouse strain
(20.2) which enabled the above result, this copy number proved to be about
80. This means that the genome of the mice in question contains some
additional 4 millions of base pairs of DNA. Also with other strains the
vector proved to be integrated in a large number of copies. For instance,
strain 40.6 has the vector in about 40 copies. The stable inheritance of
so much additional DNA has not been reported before.
However, in order to obtain a reliable picture of the mutation frequency in
different organs and tissues at low doses of the suspected agent, it is
necessary to test much more than 9,000 plaques. As stated above, a
scale-up of the phage packaging reaction by using more genomic DNA is
expensive. Take the case, for instance, that a suspected agent increases
the mutation frequency from 1 : 100,000 (spontaneous frequency) to 1 :
50,000. In theory, on the basis of the efficiency obtained one would have
to carry out a phage packaging experiment with 5 .mu.g total genomic mouse
DNA. From this one would get one mutant (one uncoloured plaque). It will
be clear that this is not sufficient in practice; for a close
determination of such a low mutation frequency at least 500,000 to 1
million of plaques will have to be viewed.
According to a preferred embodiment of the invention this problem was
solved by carrying out a pre-purification based on the idea that if the
vectors could be separated from the rest of the chromosomal DNA, the ratio
of the vector to the rest of the genome becomes much more favourable. This
means that many more vectors can be recovered with the same amount of
phage packaging extract. But the question was how to separate the
integrated vectors from the rest of the chromosomal DNA.
It was thought that if the vector, which is about 50,000 base pairs long,
has no recognition sites for a certain restriction enzyme which frequently
cuts in total genomic mammalian DNA, this gives a fragment of about 1.5
millions of base pairs after tandem integration in, e.g., 30 copies. Since
a restriction enzyme frequently cutting in mammalian DNA generates
fragments having an average size of about 5000 base pairs, such a large
fragment can be rapidly purified by means of a simple separation method.
The vector used by the inventor and colleagues contained no recognition
sites for the enzyme XbaI. It has turned out that in a number of the
available transgenic mice the vectors were integrated head-to-tail in
40-80 copies. Indeed, after cutting with XbaI a fragment of more than 1
million of base pairs proved to be easily separable from the rest of the
(fragmented) genomic DNA. It was thought that a similar principle could
also be used the other way round. Plasmids integrated in the genome (which
are not larger than, e.g., about 5000 base pairs) can be simply rescued
from total genomic DNA, provided they are flanked by restriction enzyme
recognition sites unique for the plasmid; if rare restriction enzyme
recognition sites (e.g., Not I) are applied to the left and right of the
vector, a pre-purification is possible also here on the basis, in the
present case, of precisely the small size of the plasmid-containing DNA
fragment.
An alternative for pre-purification of the vectors is to interbreed the
different mouse strains, each harbouring a vector-cluster at a different
site in their genome. The latter was not checked, but integration of
injected DNA in the mouse genome is random and it is therefore highly
unlikely that two different transgenic mice are obtained with the
vector-cluster at the same site on the same chromosome. The interbreeding
will result in a mouse strain with multiple vector clusters at different
sites in the genome. In such a "multi-locus" strain many more vectors can
be rescued from each mouse cell and therefore the rescue frequency becomes
much higher; the efficiency of rescue, which is entirely dependent on the
use of host restriction-negative E. coli strains, such as E. coli C, does
not change in this way.
Phage packaging experiments with pre-purified vector-containing genomic DNA
of both untreated and ENU-treated transgenic mice (strain 20.2) showed
that per DNA sample (about 100 .mu.g total chromosomal mouse DNA) about
1,400,000 plaques could be obtained in one experiment. (ENU is ethyl
nitrosourea).
Although the invention has been described hereabove as a process for the
detection of mutations in one or more marker genes which have been
introduced into the genome of a mammal by means of a vector, wherein the
rescue and cloning of the methylated vector and the marker genes contained
therein requires the use of a restriction negative host, the invention is
generally applicable with any process for the rescue and cloning of DNA
which may be methylated. The conventional process of cloning genomic DNA
fragments does not use completely restriction-negative hosts. As a result
thereof, the library of cloned fragments will not be complete, i.e. will
not comprise the heavily methylated parts of the mammalian genome. The
present invention solves this problem by using a restriction-negative host
for the rescue and cloning of mammalian DNA which will result in a more
complete collection of genomic DNA fragments.
In a broad sense, the invention therefore provides a process for the rescue
and cloning of a DNA fragment wherein a vector containing said DNA
fragment is multiplied in a suitable bacterial host, characterized by
using as the bacterial host, a bacterial strain which is incapable of host
restriction.
Although any bacterial species for which a suitable cloning vehicle is
available may be used, a preferred embodiment of the invention is
characterized by using as the bacterial host, an Escherichia coli strain
which is incapable of host restriction. As an example of a preferred
Escherichia coli strain which is incapable of host restriction,
Escherichia coli C strains should be mentioned.
A preferred embodiment of the present invention is characterized by using
as the vector, a bacteriophage vector which is a suitable cloning vehicle
for the bacterial host concerned, the step of in vitro packaging into
phage coats being performed by means of a phage packaging extract obtained
from a bacterial strain which is incapable of host restriction.
Another, less preferred embodiment of the invention is characterized by
using as the vector, a plasmid vector which is a suitable cloning vehicle
for the bacterial host concerned.
A preferred process for the rescue and cloning of a DNA fragment wherein a
vector containing said DNA fragment is multiplied in a suitable bacterial
host is characterized by using an Escherichia coli strain which is
incapable of host restriction as the bacterial host and a bacteriophage
lambda vector as the vector, the step of in vitro packaging into phage
coats being performed by means of a phage packaging extract obtained from
an Escherichia coli strain which is incapable of host restriction. Again,
it is preferred to use Escherichia coli C strains incapable of host
restriction as bacterial host and source of the phage packaging extract,
respectively.
The invention further provides a process for detecting mutations in one or
more marker genes introduced into a mammalian genome by means of a vector,
which process comprises isolating DNA from cells of a transgenic mammal or
from mammalian cells, recovering the vector from said DNA, multiplying the
vector in a suitable bacterial host which is deficient with respect to at
least one of the marker genes, and testing marker gene expression,
characterized by using as the bacterial host, a bacterial strain which is
incapable of host restriction. Preferably, an Escherichia coli strain
which is incapable of host restriction, such as an Escherichia coli C
strain, is used as the bacterial host.
A preferred embodiment of the present invention is characterized by using
as the vector, a bacteriophage vector which is a suitable cloning vehicle
for the bacterial host concerned, the step of in vitro packaging into
phage coats being performed by means of a phage packaging extract obtained
from a bacterial strain which is incapable of host restriction.
An alternative process according to the invention is characterized by using
as the vector, a plasmid vector which is a suitable cloning vehicle for
the bacterial host concerned.
More in particular, the invention provides a process for detecting
mutations in one or more marker genes introduced into a mammalian genome
by means of a vector, which process comprises isolating DNA from cells of
a transgenic mammal or from mammalian cells, recovering the vector from
said DNA, multiplying the vector in a suitable bacterial host which is
deficient with respect to at least one of the marker genes, and testing
marker gene expression, which process is characterized by using an
Escherichia coli strain which is incapable of host restriction as the
bacterial host and a bacteriophage lambda vector as the vector, the step
of in vitro packaging into phage coats being performed by means of a phage
packaging extract obtained from an Escherichia coli strain which is
incapable of host restriction.
Again, it is preferred to use Escherichia coli C strains incapable of host
restriction as bacterial host and source of the phage packaging extract,
respectively.
A highly preferred embodiment of the present invention is characterized in
that the DNA isolated from cells of a transgenic mammal or mammalian cells
is pre-purified by fragmenting the DNA by means of a restriction enzyme
which does not have a cutting site within the vector, separating the
resulting fragments on the basis of a suitable criterion, such as
differences in size, and collecting fragments comprising the vector,
whereafter the vector is recovered from said pre-purified DNA.
A preferred process according to the invention for detecting mutations in
one or more marker genes introduced into a mammalian genome by means of a
vector, which process comprises isolating DNA from cells of a transgenic
mammal or from mammalian cells, recovering the vector from said DNA,
multiplying the vector in a suitable bacterial host which is deficient
with respect to at least one of the marker genes, and testing marker gene
expression, is characterized in that an Escherichia coli strain which is
incapable of host restriction is used as the bacterial host, a
bacteriophage lambda vector having a size of at least 30 kb is used as the
vector, at least 3 copies of said vector are integrated in the mammalian
genome in a head-to-tail arrangement, the step of in vitro packaging into
phage coats is performed by means of a phage packaging extract obtained
from an Escherichia coli strain which is incapable of host restriction,
and the DNA isolated from cells of the transgenic mammal or the mammalian
cells is pre-purified by fragmenting the DNA by means of a restriction
enzyme, such as XbaI, which does not have a cutting site within the vector
but does have such a large number of cutting sites in the mammalian genome
that it is capable of generating fragments having an average size below 10
kb, separating the resulting fragments on the basis of their size
differences, and collecting the larger fragments comprising the vector. It
is preferred to use Escherichia coli C strains incapable of host
restriction as bacterial host and source of the phage packaging extract,
respectively. It is further preferred that at least 10 copies of the
bacteriophage lambda vector are integrated in the mammalian genome in a
head-to-tail arrangement.
An alternative process according to the invention for detecting mutations
in one or more marker genes introduced into a mammalian genome by means of
a vector, which process comprises isolating DNA from cells of a transgenic
mammal or from mammalian cells, recovering the vector from said DNA,
multiplying the vector in a suitable bacterial host which is deficient
with respect to at least one of the marker genes, and testing marker gene
expression, is characterized in that an Escherichia coli strain which is
incapable of host restriction is used as the bacterial host, the vector
integrated in the mammalian genome is a plasmid vector flanked by
restriction enzyme cutting sites which do not occur within the vector, and
the vector is recovered by fragmenting the DNA isolated from cells of the
transgenic mammal or the mammalian cells by means of said restriction
enzyme and ring closure. As an example thereof, the invention provides a
process for detecting mutations in one or more marker genes introduced
into a mammalian genome by means of a vector, which process comprises
isolating DNA from cells of the transgenic mammal or from mammalian cells,
recovering the vector from said DNA, multiplying the vector in a suitable
bacterial host which is deficient with respect to at least one of the
marker genes, and testing marker gene expression, which process is
characterized in that an Escherichia coli strain which is incapable of
host restriction is used as the bacterial host, the vector integrated in
the mammalian genome is a plasmid vector having a size of not more than 10
kb flanked by restriction enzyme cutting sites which do not occur within
the vector and occur in such a small number in the mammalian genome for
being capable of generating fragments having an average size above 20 kb,
such as NotI restriction sites, and the DNA isolated from cells of the
transgenic mammal or the mammalian cells is pre-purified by fragmenting
the DNA by means of said restriction enzyme, separating the resulting
fragments on the basis of their size differences and collecting the
smaller fragments comprising the vector.
The invention can be directly applied in the efficient cloning of
methylated DNA by the use of bacteriophage lambda vectors as cloning
vehicles, packaging extracts derived from host restriction-negative E.
coli strains, such as E. coli C and the same or comparable E. coli strains
for plating. A second major application follows automatically and involves
the testing of suspected agents for carcinogenicity. Shortly after
treatment (e.g., after a few hours or a few days) the effect of the
suspected agent, in terms of mutation frequency, can be directly studied
in DNA from different organs and tissues of the treated transgenic
animals, such as mice. Highly qualified staff is not required for this.
Besides, the nature of the mutations can be determined in a relatively
simple manner, because in this system the mutated lacZ gene is already
cloned. When studying endogenous genes, such HPRT, it is always necessary
first to obtain the mutated gene in pure condition. In the present case,
each plaque is a small reservoir of lacZ genes which can be easily
cultured further. The nucleotide sequence of the lacZ gene can now
directly be determined by means of classical methods.
Besides the Ames test and the chemical structure of the suspected agent,
the transgenic mouse model here described could well be a so desired third
(in vivo) test (for a discussion of this problem, see Ashby and Tennant,
1988 and Hay, 1988). The introduction of the present invention as third
test saves expenses, because it will then be necessary less often to make
use of prolonged animal tests and of expensive veterinary pathologists.
Introduction thereof also implies fewer inconveniences for the animals
used, while the number of animals can be strongly reduced. All these
factors make the present invention a suitable candidate for a third
short-term test for chemicals suspected of carcinogenicity.
An important third field of application related to the second is the use of
the above described transgenic mouse model for testing the potentially
protective effect of certain agents. Although the prevention of
unnecessary exposure to potentially carcinogenic agents is of course
always preferred to protective steps, circumstances making said exposure
inevitable are imaginable indeed. A simple example is the deliberate
exposure of repair crews to much too high doses of carcinogenic agents in
case of industrial accidents (or radiation in, e.g., accidents with
nuclear power plants). The availability of pharmaceutics having a
protective effect against injury to the heritable material could bring
relief here. However, also apart from this type of extreme conditions,
injury to the heritable material is not always avoidable. For instance,
there are indications that in many houses the amount of background
radiation is extremely high, which makes protection desirable. On the
other hand, in some latitudes the amount of ultraviolet light is so high
that the risk of cutaneous cancer considerably increases. In a more
general sense, it may be said that exposure to (unduly) high doses of
DNA-injuring factors will increasingly become an inevitable reality of
everyday life. In this connection it is relevant to aim at protection,
e.g., by adding protective agents to the diet. Although it is suggested
that certain diet factors, such as vitamin c, SOD (superoxide dismutase),
carotene, etc. would indeed protect against DNA-injuring agent | | |
