 1997 Oxford University Press
Human Molecular Genetics, 1997, Vol. 6, No. 8 1375–1382
Human mini-chromosomes in mouse embryonal
stem cells
M. H. Shen, J. Yang, M.-L. Loupart1, A. Smith1 and W. Brown*
Cancer Research Campaign Chromosome Molecular Biology Group, Biochemistry Department, Oxford
University, South Parks Road, Oxford OX1 3QU, UK and 1Centre for Genome Research, University of Edinburgh,
King’s Buildings, West Mains Road, Edinburgh EH9 3JQ, UK
Received April 23, 1997; Revised and Accepted May 28, 1997
We have introduced human mini-chromosomes of 4 Mb
and ∼15 Mb in size into mouse embryonal stem cells.
Although these human mini-chromosomes are stable in
hamster and chicken cells, they re-arrange or segregate
aberrantly in the embryonal stem cells and are rapidly
lost in the absence of selection. However, one of the
mini-chromosomes re-arranged, acquired mouse
centromeric sequences and was then stably maintained
for at least 60 population doublings in culture. This
mini-chromosome, which is 4 Mb in size, is a candidate
for a mouse germ line chromosome vector.
mini-chromosomes will be practical. Here we have addressed the
second problem by introducing mini-chromosomes derived from
the human Y chromosome into mouse ES cells and then studying
their stability. Our results demonstrate that these human
mini-chromosomes are exceptionally unstable in mouse ES cells
and are lost as often as once in every 15 cell divisions. In situ
hybridization to intact cells indicates that inaccurate segregation
at mitosis causes the instability. However, we describe a
mini-chromosome of human origin which has acquired mouse
centromeric sequences by re-arrangement in the ES cells and
which is stable in ES cells for at least 60 population doublings.
INTRODUCTION
Introduction of human mini-chromosomes into mouse
ES cells
There is currently interest in the possibility of engineering
mammalian artificial chromosome vectors (MACs) for the mouse
germ line. It has been speculated that they may allow new
approaches to genome engineering (1), to the study of gene
expression and to the understanding of the molecular basis of
chromosome maintenance and segregation in both germ line and
somatic cells. Artificial chromosomes have also been suggested
as models of vectors that might ultimately be used for therapeutic
purposes (2). There are two approaches to building MACs; in the
bottom up approach they are assembled in vitro from cis-acting
functional sequences. In the top down approach they are
engineered in vivo by the systematic modification of minichromosomes generated by telomere directed chromosome
breakage.
The top down approach is being applied to both the human X
and Y chromosomes (3,4) and has generated mini-chromosomes
between 2.5 and 8 Mb in size (1,3,4) with mini-chromosomes as
small as 4 Mb being mitotically stable for several months in
culture (4). The top down approach to developing a practical
vector system requires first of all the ability to modify
mini-chromosomes efficiently and then to successfully introduce
the mini-chromosomes into the mouse germ line. The
demonstration that human chromosomes can be introduced into
the chicken lymphoid cell line DT40 (5) by microcell fusion,
modified by homologous recombination at efficiencies of
between 1 and 30% and then returned to human cells for
functional analysis (6) suggests that efficient modification of
RESULTS
∆∆2 is a 4 Mb mini-chromosome which was derived from the
long arm of the human Y chromosome by three rounds of
telomere directed chromosome breakage (4) and which is
mitotically and structurally stable in CHO cells. The human DNA
in this mini-chromosome is re-arranged with respect to the
starting Y chromosome in several respects (details of the mapping
will be presented elsewhere). Most importantly the alphoid DNA
which is normally found as a single array at the centromere of the
Y is broken into three arrays (see legend to Fig. 1 for details). ∆∆2
is marked with a hygromycin resistance thymidine kinase fusion
gene with a cytomegalovirus promoter (7) which was readily
selectable in mouse ES cells. This made it practical to use ∆∆2 to
determine whether it would be possible to move
mini-chromosomes into ES cells and to determine how stable
they were.
We used microcell fusion to transfer ∆∆2 from CHO cells into
ES cells of the CGR8 line (8). After four fusions we isolated a
total of 22 hygromycin resistant colonies. We analysed these by
pulsed field gel electrophoresis of uncut DNA (Fig. 1B, C) and
demonstrated that 21 contained autonomous mini-chromosomes,
20 of which were detected by filter hybridization with an alphoid
DNA probe and one (mini-chromosome 310) by a total human
DNA probe only (not shown). One of these clones, 324, contained
two mini-chromosomes and was subcloned to yield S18 (1.6 Mb)
and S19 (3.5 Mb). We also analysed the microcell hybrids by
fluorescent in situ hybridization to metaphase chromosomes and
*To whom correspondence should be addressed. Tel: +44 1865 275225; Fax: +44 1865 275259; Email: [email protected]
1376 Human Molecular Genetics, 1997, Vol. 6, No. 8
Figure 1. (A) Physical map of mini-chromosome ∆∆2. The map of ∆∆2 was derived by restriction enzyme digestion, pulsed field gel electrophoresis and filter
hybridization. Details of the mapping will be described elsewhere. The alphoid DNA on this chromosome is present on three arrays; the smallest of which is 11.7 kb
in size and resides on a 20 kb BclI fragment adjacent to the neo gene at the left end of the mini-chromosome; the second array of alphoid DNA is located on a 90 kb
BclI fragment which is 50 kb from the neo gene at the left end of the chromosome and the third array is located on a 100 kb BclI fragment which is 2.5 Mb from the
neo gene. Interrupting the 90 kb and 100 kb alphoid arrays is a 1.5 Mb array of DNA of DYZ1 and DYZ2 sequences and a stretch of euchromatic DNA derived from
interval 6 (9) of the long arm of the human Y chromosome which includes the STSs sY129–143. The DNA between the 100 kb array of alphoid DNA and the
hygromycin resistance-thymidine kinase fusion gene at the right end of the mini-chromosome is derived from interval 5 of the long arm of the Y chromosome (9),
includes STSs sY88, 90, 106 and 109 and a 30 kb block of DYZ1 sequences. The positions of the two larger alphoid arrays and of the DYZ1, DYZ2 array are indicated.
(B and C) Pulsed field gel electrophoresis of ∆∆2 derived mini-chromosomes. DNA extracted from either the original CHO hybrid containing ∆∆2, from the ES cell
line CGR8 or from 22 hybrid cells derived by the introduction of ∆∆2 into ES cells was size fractionated by pulsed field gel electrophoresis and then analysed by filter
hybridization with an alphoid DNA probe. (D) DYZI content of mini-chromosomes derived by transfer of ∆∆2 into ES cells. DNA extracted from mini-chromosome
containing ES cells was digested with HindIII, size fractionated by pulsed field gel electrophoresis and then analysed by filter hybridization with a DYZI probe. (E)
Alphoid DNA content of mini-chromosomes derived by transfer of ∆∆2 into ES cells. DNA extracted from mini-chromosome containing ES cells was digested with
BclI, size fractionated by pulsed field gel electrophoresis and then analysed by filter hybridization with an alphoid DNA probe. (F) Fluorescent in situ hybridization
of alphoid DNA to metaphase chromosomes of clone S18 containing a 1.6 Mb mini-chromosome. In addition to the S18 mini-chromosome this line had a 41, XY
karyotype and included a metacentric chromosome 11.
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Figure 2. (A) Stability of mini-chromosome S18 in ES cells analysed by
fluoresence in situ hybridization to metaphase chromosomes with an alphoid
DNA probe. Circles refer to autonomous chromosomes while the squares refer
to translocated chromosomes. The filled symbols represent the respective
samples in the presence of selection while the open symbols represent the
respective samples in the absence of selection. (B) Stability of mini-chromosome
∆Yq2.5 in ES cells analysed by fluoresence in situ hybridization to metaphase
chromosomes with an alphoid DNA probe. The filled circles represent the
percentage of metaphase cells containing autonomous mini-chromosomes in the
presence of selection while the open circles represent the percentage of cells
containing autonomous mini-chromosomes in the absence of selection.
21 of the 22 were also found to contain autonomous
mini-chromosomes with either an alphoid or human DNA probe.
An example of this sort of result is shown in Fig. 1F for the
mini-chromosome containing clone S18. This mini-chromosome
was detected in 70% of the spreads at an average copy number of
one. The sizes of the mini-chromosomes detectable in the ES cells
usually differed from the starting chromosome ∆∆2 (Fig. 1B, C)
suggesting that ∆∆2 had been re-arranged at or after transfer into
the ES cells. Microcell transfer is often associated with
chromosome re-arrangement but the level detected in these
experiments is atypically high. For example 50% of the clones
derived in a transfer of the short arm mini-chromosome ∆1 from
CHO cells into the human fibroblast line HT1080 contained intact
mini-chromosomes (9).
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We wished to analyse the re-arrangements of the ∆∆2 derived
chromosomes further and therefore picked 14 clones for detailed
study. We digested DNA from each with either BclI which does
not cut within the arrays of alphoid DNA or with HindIII which
does not cut within the DYZ1,2 array and then analysed the
digests by filter hybridization either with a DYZ1 probe (Fig. 1D)
or with an alphoid DNA probe (Fig. 1E). We also analysed the
clones for the Y derived STSs present on ∆∆2 (Table 1). These
results demonstrated that all of the chromosomes were deleted for
human DNA and that, with the exception of clone 311, these
deletions fell into a single nested set extending from the
unselected neo gene at the left end of the mini-chromosome. Two
results are particularly important; firstly 11 of the 14 clones retain
the 100 kb array of alphoid DNA and secondly, while there is no
simple relationship between the sizes of the mini-chromosomes
and the amount of human DNA that has been deleted, the smallest
mini-chromosomes (S18 and 342) are 1.6 Mb in length which is
only 100 kb larger than the distance from the HyTk gene to the
100 kb array of alphoid DNA. These results can be explained if
∆∆2 is re-arranging mainly as a result of breakage between the
two arrays of alphoid DNA. Such breakage would occur if each
alphoid array functioned as an independent centromere upon
introduction into the ES cells and would result in two fragments
only one of which would retain the HyTk marker gene under
selection. These fragments would then enter a series of chromatid
type breakage fusion bridge cycles (10) until either healed with
a mouse telomere or lost from the cell. The chromosomes in
clones 11, 310 and 311 have structures that appear inconsistent
with this hypothesis and will be considered further in the
Discussion.
The re-arrangements of ∆∆2 in ES cells may have been a
consequence of the fact that the chromosome contained a
re-arranged centromere and so we set out to introduce a derivative
of the Y chromosome containing just a single array of alphoid
DNA into ES cells. We therefore used a random telomere
breakage construct tagged with a hisD gene (conferring resistance
to histidinol) driven by a chicken actin promoter to break the
intact human Y chromosome in the CHO hybrid cell line 853.
Table 1. The STS and sequence content of the ∆∆2 derived mini-chromosomes in the ES cell line CGR8
nd, not done; r, re-arranged. Clone 11 contains two re-arranged arrays of alphoid DNA, the origin of which is uncertain but which the sequence content of the
chromosome suggests derive from the 100 kb array of alphoid DNA. This uncertainty is indicated by a ? in the column of the clone 11 row corresponding to the
90 kb array of alphoid DNA and an r in the clone 11 row corresponding to the 100 kb array of alphoid DNA.
1378 Human Molecular Genetics, 1997, Vol. 6, No. 8
Figure 3. ∆Yq2.5 segregation in ES cells analysed by fluorescent in situ hybridization to intact anaphase cells. (A) The figure includes two mitotically dividing cells
at anaphase. The cell on the left has a short spindle and it is not possible to be clear whether the mini-chromosomes are lagging with respect to the mouse centromeres.
However, in the cell on the right the lower set of chromosomes includes a sister mini-chromosome ∆Yq2.5 which is lagging. (B) Accurate 1:1 segregation of
mini-chromosome ∆Yq2.5 in ES cells analysed by fluorescent in situ hybridization to intact anaphase cells.
Figure 4. Stability of mini-chromosome S18 in DT40 cells analysed by pulsed
field gel electrophoresis. Mini-chromosome S18 was transferred from ES cells
into DT40 cells by microcell fusion. The size and STS content (Table 1) of the
mini-chromosome was unaltered by the transfer. DNA extracted from the
starting clone S18/ES, from the initial DT40 microcell hybrid S18/DT40 and
from DT40 hybrids containing S18 after 30 and 60 generations in the absence
or presence of selection was analysed by pulsed field gel electrophoresis (2.2
V/cm, 0.75% agarose, 3.5C, 8 min pulse time, 72 h run).
This led to the isolation of the mini-chromosome ∆Yq2.5 which
was broken on the long arm between sY94 and sY98 in interval
5 (11) and thereby deleted for the long arm heterochromatin.
Restriction analysis of the DYZ3 and DYZ5 arrays and STS
analysis established that the mini-chromosome was a simple
truncation derivative of the intact Y chromosome and was not
otherwise re-arranged (not shown). Although this minichromosome cannot be resolved by pulsed field gel
electrophoresis we estimate from the 60 Mb size of the intact Y
chromosome (11) that this mini-chromosome is ∼15 Mb in
length. We introduced ∆Yq2.5 into ES cells of the CGR8 line and
isolated five histidinol resistant clones. Structural analysis
indicated that this mini-chromosome re-arranged much less upon
transfer into ES cells than ∆∆2; L1 fingerprinting of an XbaI
digest indicated that one clone contained a re-arrangement.
Similarly one other clone contained an extra cognate DYZ3
(alphoid) fragment. All five clones, however, contained the
complete set of Y STSs present on the starting ∆Yq2.5 and an
autonomous mini-chromosome that was cytogenetically
indistinguishable from the ∆Yq2.5 in the CHO cells (not shown).
The mini-chromosome ∆Yq2.5 is too large to be size fractionated
by pulsed field gel electrophoresis and therefore it is not possible
to conclude that the three of the five clones containing no
detectable re-arrangement are intact but any re-arrangements are
likely to be small. These results indicated that transfer of intact
mini-chromosomes from somatic cells into ES cells is possible
and that the re-arrangements seen in the ∆∆2 transfers are
probably a consequence of the behaviour of the re-arranged
centromere on this chromosome in the mouse ES cells.
Human centromeres function poorly in mouse ES cells
Stable inheritance at cell division would be necessary for some
applications of mini-chromosome based vectors and so we set out
to measure the stability of the mini-chromosomes derived from
∆∆2 and of mini-chromosome ∆Yq2.5 in ES cells. We measured
the mitotic and structural stabilities of the mini-chromosomes by
cytogenetic methods, by gel electrophoresis of intact DNA and by
measuring the retention of mini-chromosomal sequences by
restriction enzyme digestion, gel electrophoresis and filter
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hybridization. The results of the three methods were consistent
with one another and demonstrated that all with one exception
(mini-chromosome 341) were unstable. The stability of the ∆∆2
derived mini-chromosome S18 (Fig. 2A) is illustrated as an
example. In the absence of selection S18 was lost about once
every 15 generations. In the presence of selection the autonomous
mini-chromosome was lost and mini-chromosomal DNA was
observed to be translocated onto a mouse chromosome. For all but
one of the ∆∆2 derived mini-chromosomes that we studied the
kinetics of loss in the absence of selection were approximately first
order and all were so rapidly lost that by 30 generations in the
absence of selection less than 15% of the cells contained
autonomous mini-chromosomes. It seemed possible that the
instability that we detected in the ∆∆2 derived mini-chromosomes
was a consequence of the fact that they were re-arranged and so we
also measured the stability of mini-chromosome ∆Yq2.5 in ES
cells. This mini-chromosome was also unstable (Fig. 2B). The
kinetics of loss were not first order but by 60 population doublings
only 15% of the cells retained the mini-chromosome. These results
therefore established that mini-chromosomes with human
centromeres are unstable in ES cells.
We wanted to know whether the mini-chromosomes were being
lost as a result of inaccurate segregation and so we used in situ
hybridization of human genomic DNA to detect the presence of the
human mini-chromosome ∆Yq2.5 in intact dividing cells. These
studies were consistent with the view that the mini-chromosomes
were segregating abnormally; we examined a total of 444 anaphase
cells and in 21 one or other of the sister mini-chromosomes was
observed to lag with respect to the centromeres of the mouse
chromosomes. An example of this data is illustrated in Figure 3.
Figure 3B illustrates an anaphase cell in which the mini-chromosomes are segregating normally. Figure 3A includes two mitotically dividing cells at anaphase. The cell on the left has a very short
spindle and although the human mini-chromosome has a position
in each half of the spindle that is consistent with the positions of the
mouse centromeres it is not possible to be sure whether it is
segregating normally. In the dividing cell to the right, however, the
mini-chromosome in the lower daughter is clearly segregating
aberrantly and lags with respect to the mouse centromeres.
The observation that human mini-chromosomes are mitotically
unstable in mouse ES cells would be unremarkable were it the case
that chromosomes of one mammalian species are generally
unstable in the cells of another mammalian species. That this is not
so is suggested by the observation that human mini-chromosomes
are stable in Chinese hamster cells (3,4) for at least one hundred
population doublings in the absence of any applied selection.
However, in order to explore further the question of whether mouse
cells are exceptional and chromosomes of one vertebrate species
are generally stable in cells of another we transferred the ∆∆2
derived mini-chromosome, S18 from ES cells into DT40 cells and
the mini-chromosome ∆Yq2.5 from CHO cells into chicken DT 40
cells. We chose the DT40 line for two reasons; firstly it is not a
mammalian cell and if the mini-chromosomes were stable in this
line then it would emphasize the unusual characteristic of the
mouse ES cells and secondly DT40 allows efficient modification
of human mini-chromosomes by sequence targeting approaches.
The results of these experiments demonstrated that both minichromosomes were stable in the DT40 cells. Figure 4 shows a gel
analysis of the stability of S18 in DT40 cells in the absence and
presence of selection (Table 1). We also analysed the stability of
S18 in DT40 cells by cytogenetic methods and this showed that
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Figure 5. (A) Stability of mini-chromosome 341 analysed by in situ hybridization
to metaphase chromosomes. Circles refer to autonomous chromosomes while the
squares refer to translocated chromosomes. The filled symbols represent the
respective samples in the presence of selection while the open symbols represent
the respective samples in the absence of selection. (B) Stability of mini-chromosome 341 analysed by pulsed field gel electrophoresis. DNA extracted from ES cell
clone 341 after the indicated time in culture in either the absence or presence of
selection was size fractionated by pulsed field gel electrophoresis (0.6% agarose,
0.75 V/cm, 1 h pulse time, 3.5C, 11 days run time), blotted and hybridized with
an alphoid DNA probe. The arrow indicates the position of the 341d (341 derived)
mini-chromosome. (C) Reselection and stability of mini-chromosome 341d
analysed by pulsed field gel electrophoresis. DNA extracted from the ES cell clone
341 was size fractionated by pulsed field gel electrophoresis. The ES 341 cells were
isolated either immediately after microcell transfer, at the start of the experiment
designed to measure stability (t0), after 60 generations in the absence of selection
t60–, after 60 generations in the absence of selection followed by 14 generations in
the presence of selection t60–14+, and after a further 30 or 60 generations in the
absence of selection; t60–14+30– and t60–14+60–. (D) Stability of mini-chromosome
341d analysed by in situ hybridization to metaphase chromosomes. The open circles
refer to autonomous chromosomes maintained in the absence of selection.
after 60 generations in the absence of selection the mini-chromosome was quantitatively retained at one copy per cell. Similarly
∆Yq2.5 was quantitatively retained at one copy per cell in DT40
cells after 60 generations in the absence of selection (not shown).
These results demonstrate that the instability of mini-chromosomes
in mouse ES cells is unusual.
Isolation of mini-chromosome containing a mouse
centromere that is stable in ES cells
One of the ∆∆2 derived mini-chromosomes showed anomalous
kinetics of chromosome loss when analysed cytogenetically with
30% of the cells containing an autonomous mini-chromosome
(Fig. 5A) after 60 generations in culture. Gel electrophoresis of
uncut DNA revealed the presence of a novel 4 Mb mini-chromo-
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Figure 6. Mini-chromosome 341d contains both mouse major and minor satellite DNA. Metaphase chromosomes from 341 cells isolated after 60 generations in the
absence of selection and a further 14 generations in the presence of selection were hybridized in situ with: (A) a mixture of alphoid DNA (fluorescein detection) and
mouse major satellite DNA (texas red detection) and counter stained with DAPI (blue); (B) a mixture of alphoid DNA (fluorescein detection) and mouse minor satellite
DNA (texas red detection) and counter stained with DAPI (blue).
some 341d that was present in the cells either in the absence or
presence of selection (Fig. 5B). This mini-chromosome was
larger in size than the predominant mini-chromosome in the
starting culture which was 2.5 Mb in size. Gel electrophoresis and
filter hybridization demonstrated that the mini-chromosome had
lost part of the alphoid DNA because the hybridization signal with
this probe was weaker for the 4 Mb mini-chromosome than for the
2.5 Mb mini-chromosome which originally predominated in the
population and this was confirmed by restriction analysis (not
shown). In order to better analyse the properties of this
chromosome we re-applied selection to the culture that had been
grown in its absence. After 7 days 80% of the cells contained the
4 Mb mini-chromosome detectable with an alphoid DNA probe.
The only human DNA present in these cells was present on the
stable mini-chromosome 341d. Mini-chromosome 341d was then
stably maintained for a further 60 generations in the absence of
applied selection (Fig. 5C, D). The stability of this mini-chromosome suggested that it had a functioning centromere and so we
wanted to know whether this 4 Mb mini-chromosome had
acquired mouse centromeric sequences. Therefore we hybridized
metaphase chromosomes with alphoid DNA and with either
mouse minor or mouse major satellite sequences labelled with
digoxygenin or biotin respectively. The results indicated that the
mini-chromosome 341d included alphoid DNA, minor satellite
DNA and major satellite DNA (Fig. 6A, B). The mouse
centromeric sequences were absent from the mini-chromosome
which predominated in the original culture (not shown). Together
these results indicated that 341d had acquired a mouse centromere
and was thereby stabilized.
DISCUSSION
We have demonstrated that it is possible to introduce gel
resolvable mini-chromosomes into mouse embryonic stem cells
but that in general they are unstable because they segregate
inaccurately at mitosis. The instability of these human minichromosomes in ES cells is, however, not a reflection of a general
rule that chromosomes of one vertebrate species are unstable in
cells of another because human mini-chromosomes are stable in
hamster cells (3,4) and as we have shown here, they are also stable
in chicken cells. It is well known that human chromosomes are
unstable in hybrids generated by fusion with mouse cells (13) and
our results suggest that this instability may have its origin in,
amongst other things, inaccurate segregation. In this context it is
important to note three of our results which suggest that
inaccurate segregation is a consequence of poor rather than no
centromere function. Firstly, the pattern of re-arrangement of the
∆∆2 mini-chromosome is consistent with the idea that the alphoid
DNA is functioning as a centromere in the ES cells. Secondly,
although the ∆∆2 derived mini-chromosomes are frequently
observed to translocate onto a mouse chromosome they are
maintained at about one copy per cell when under selection.
Thirdly, they are lost at a rate of less than once every fifteen
divisions when selection is relaxed. To our knowledge cell type
specific centromere function has not been observed before in any
mammalian cell type and thus while the behaviour of the human
mini-chromosomes in ES cells is a setback for vector development it creates new possibilities for the study of centromere
function. It may, for example, be possible to use human
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mini-chromosomes as vectors to develop functional assays for
mouse centromeric DNA. Mouse minor satellite DNA is a
candidate for the functional centromeric DNA and it should be
easy to add minor satellite DNA to a mini-chromosome by
targeting in the DT40 cell line and then determine whether this
improves mini-chromosome stability after transfer into ES cells.
Similarly it may be possible to use ES cells to identify
centromeric DNA binding proteins necessary for full centromere
function. If a single protein is limiting then this protein must be
a DNA binding protein and have a lower affinity for human
centromeric DNA than for mouse centromeric DNA. CENP-A is
a candidate.
We have used two types of human mini-chromosome in our
experiments. The mini-chromosome ∆Yq2.5 is a simple derivative of the human Y chromosome generated by truncation of the
long arm which contains a single centromeric array of alphoid
DNA and the entire short arm. This mini-chromosome which we
estimate to be ∼15 Mb in length re-arranges very little after
microcell transfer into ES cells. ∆∆2 is a mini-chromosome
derived from the long arm of the human Y chromosome by three
rounds of telomere directed chromosome breakage in CHO cells
(4) and contains two arrays of alphoid DNA each of ∼100 kb in
size and a smaller array of 11 kb in size. Three of the ∆∆2 derived
ES cell mini-chromosomes (mini-chromosomes 11, 310 and 311)
have structures that appear to be inconsistent with the explanation
that ∆∆2 was re-arranging in ES cells simply as a consequence of
the two larger arrays of alphoid DNA functioning as independent
centromeres. Mini-chromosome 11 contains a re-arranged block
of alphoid DNA which is suggested by the deletion map to arise
from the 100 kb array. The origin of this re-arrangement is not
known but it could have arisen either during or shortly after the
microcell transfer procedure. Mini-chromosome 310 lacks
detectable alphoid DNA and this raises the question of how this
mini-chromosome could have been generated and what if any
sequence is functioning as a centromere on this
mini-chromosome. One possibility is that mini-chromosome 310
could have arisen from an alphoid containing breakage product
if exonuclease digestion had removed alphoid DNA prior to the
healing event necessary to stabilize the mini-chromosome.
Chromosome 310 was present as a single autonomous copy in
45% of the cells and at two copies in 5% of the cells at the start
of the stability studies but by 30 generations of culture in the
absence of selection it was completely lost. These data are not
strong enough to indicate whether this mini-chromosome is any
less stable than other ∆∆2 derived mini-chromosomes but the fact
that mini-chromosome 310 is present at only one copy per cell in
almost half the cells analysed at the start of the experiment
suggests that it possesses a centromere. The identity of the
centromere on this chromosome, however, remains to be
identified. Mini-chromosome 311 has a structure that could have
arisen if the products of two different breakage events had fused
to one another. The structural stability of ∆∆2 in CHO cells (4) as
compared with the structural instability of ∆∆2 in ES cells raises
the question of what sequences are functioning as a centromere
on this chromosome in CHO cells. We are addressing this
problem in other work.
Recently there has been important progress in MAC
development with the demonstration that autonomous minichromosomes are formed de novo when cloned alphoid DNA
together with undefined genomic DNA and human telomeric
sequences is introduced into human HT1080 fibroblasts (15).
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This approach differs from the one described in this paper in that
accurately segregating chromosomes are apparently formed de
novo. Success in assembling and manipulating a defined structure
by either of the two approaches, however, requires knowledge of
the size and sequence requirements for mammalian chromosome
function, understanding of the behaviour of centromeric DNA in
different cell types and the ability to manipulate efficiently
chromosome structure. In all of these areas the work described
here represents significant progress. We have shown that we can
move mini-chromosomes into ES cells, we can recover them into
the chicken lymphoid line DT40 and most importantly we have
identified a mini-chromosome, 341d, which contains a mouse
centromere and is stably maintained in ES cells for 60 generations
in the absence of selection. Further work will be aimed at
determining the structure of 341d and determining how stably it
is maintained in developing animals.
MATERIALS AND METHODS
Cell culture and microcell fusion
CGR8 embryonal stem cells were cultured and maintained as
described in Smith (12). Microcell fusion between CHO donors
and CGR8 recipients was carried out as follows. Four 15 cm
dishes of mini-chromosome containing CHO (hprt–, hygromycin
or histidinol resistant) cells were grown to ∼70% confluence and
then colcemid was added to 0.1 µg/ml. The cells were cultured for
another 24 h, harvested and then resuspended in 40 ml of a
pre-warmed 1:1 mix of Percoll (Pharmacia) and serum free
Dulbecco’s modified Eagle’s medium (DMEM) containing
Cytochalasin B at a final concentration 10 µg/ml. The suspension
was centrifuged in a prewarmed Beckman JA20 rotor at 19 000
r.p.m. for 75 min at 37C. Microcells, distributed in two visible
bands, were harvested by aspiration and then collected by
centrifugation at 1500 g for 10 min in a swinging bucket rotor,
washed once in DMEM and then resupended in 10 ml of serum
free DMEM. CGR8 ES cells (hprt+, hygromycin and histidinol
sensitive) were grown to ∼70% confluence, harvested and
counted. Between 5 × 107 and 108 ES cells were mixed with the
10 ml of microcells, pelleted, resuspended in 1 ml serum free
DMEM and left for 10 min at room temperature. The mixture was
then pelleted, resuspended in 1 ml 50% polyethylene glycol
(Boehringer Mannheim) and incubated at room temperature for
90 s. Ten ml of serum free DMEM was added and the mixture was
left at room temperature for a further 30 min. The fusion was then
washed twice in serum free Glasgow modified Eagle’s medium
(GMEM) and then resuspended in GMEM supplemented with
10% foetal calf serum and leukaemia inhibiting factor/
differentiating inhibitory activity (12). The cells were plated out
onto ten 15 cm Petri dishes and cultured overnight before adding
selective medium which was hypoxanthine aminiopterin and
thymidine togther with either hygromycin at 100 µg/ml or
histidinol at 0.8 mg/ml. Colonies were picked after 8–10 days.
Microcell fusion between ES donors and DT40 recipients was as
above except that the ES cells were cultured for 9–10 h in
colcemid at 0.08 µg/ml before enucleation. The ratios of cell
numbers were similar to those used in the fusion between CHO
derived microcells and ES cells. The fused cells were cultured
overnight to allow contaminating intact ES cells to attach,
unattached cells, mainly DT40, were then harvested, plated into
10–15 96 well microtitre plates and selection was applied using
1382 Human Molecular Genetics, 1997, Vol. 6, No. 8
hygromycin at 2 mg/ml. Clones were picked after two weeks. In
order to isolate the mini-chromosome ∆Yq2.5 we transfected the
Chinese hamster human hybrid line 853 with a telomere breakage
construct tagged with a Salmonella typhimurium hisD gene
driven by a chicken β-actin promoter and screened ∼8000 stably
transfected cells by colony hybridization with the short arm probe
DXYS20 and the long arm probe DYZ1 with the intention of
isolating a Y chromosome derivative truncated on one or other of
the two arms (14). After three rounds of sub-cloning we isolated
one mini-chromosome, ∆Yq2.5, which was broken on the long
arm between sY94 and sY98 in interval 5 (11) of the long arm and
was deleted for the long arm heterochromatin.
58, 62, 63, 68, 76, 82, 84, 85, 94, 98, 100, 135, 140, 150, 151, 159,
160 and 182.
ACKNOWLEDGEMENTS
We thank Tatsuo Fukagawa and Peter Corish for advice and
encouragement. We also thank Lyndal Kearney and Veronica
Buckle for the use of microscopes, Ted Evans for cytogenetic
advice and Peter Shaw for the images in Figure 3. The work was
supported by the Wellcome Trust (M-LL, MHS), the Cancer
Research Campaign (JY, WB) and the BBSRC (AS).
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