Our efforts under the RTN are focussed on generating artificial chromosomes
which will function in mouse cells. Although the current alphoid DNA based
chromosomes form with reasonable efficiency in human cells (Ebersole et al., 2000;
Ikeno et al., 1998) and express genes which they contain (Grimes, Larin) in the
absence of selection, equivalent mouse artifical chromosomes do not exist. They are
needed for two principal reasons. First the mouse system is the only mammalian
system currently available in which the germ line can be manipulated. If we wish to
test the functionality of artificial chromosomes in male and female meiosis these
chromosomes need to work in the mouse. Secondly a whole animal system is
necessary to test the effect of incorporation of genes with tissue specific expression
patterns into artificial chromosomes to confirm that these expression patterns are not
altered.
We have taken two approaches to this problem. In the first, we have
attempted to transfer existing alphoid DNA based artificial chromosomes from human
cells to mouse LA9 cells by microcell fusion. This has not been successful despite
multiple attempts and we can not distinguish between the inability of these
chromosomes to function mitotically in mouse cells and technical problems because
difficulties in preparing microcells and fusing them to the mouse cell line. An
independent approach is to exploit a chromosome generated by telomere truncation
of a human Y derived chromosome ST1. This chromosome has been passaged
through a number of cell lines including mouse LA9 cells in which it segregates
correctly, and the chicken line DT40. The chromosome has acquired mouse DNA in
this process and our working hypothesis is that these sequence should be capable of
providing the basis for a murine artificial chromosome.
To effect this we have started to construct a BAC library from the ST1
chromosome in a chicken DT40 host background. The initial approach was to define
fragments containing human alphoid DNA and mouse minor (and major) satellite
DNA in a number of restriction digests which were compatible with PAC vectors.
Fragments of 145 kb containing minor satellite were cloned from gel purified material.
Bacterial colonies were screened with C57 (1.2 Kb) minor satellite probe. Screening
allowed the isolation of 16 positive clones. Complete Not I digestion was performed
and restricted DNA was separated by PFGE. Surprisingly, all the positive
recombinants showed a similar digestion pattern, consisting of four Not I fragment of
approximately 4 Kb, 3.5 Kb 2 Kb and 0.6 Kb. All the clones were estimated to be
approximately 370 Kb. Only clone 106 ( 290 Kb) digested with Not I released two
fragment of approximately 5 Kb and 0.9 Kb. Further investigations demonstrated that
all these clone derived from a large scale amplification process occurred during the
growth of bacterial cells
To check the stability and the presence of minor satellite in the above clones, we
proceeded with a second screening. This investigation showed that minor satellite
was still present only in few clones ( clones 99, 100, 105, 106).
Three different minilibraries were constructed starting from 200 Kb-350 Kb-500 Kbsize fractioned DT40 St1 DNA. Screening of the above libraries, using C57 minor
satellite probe, permitted the isolation of 6 clones from 200 Kb minilibrary, 8 clones
from 350 Kb minilibrary, and 1 clone from 500 Kb minilibrary. All these clones
resulted negative at the second screening.
Coupled with previous experience these results suggest that it is not easy to
generate minor satellite containing PACs which faithfully reflect the DNA sequence in
the ST1 chromosome. Nevertheless the clones which we have contain telomere and
minor satellite sequences and therefore have the essential components required for
chromosome formation. We have lipofected clones 105 and 106 into HT1080 cells
and are currently analysing clones selected using the blasticidin resistance marker.
We are exploring ways to surmount the problems of cloning candidate
centomeric repetitive sequences in bacterial hosts. We will test a concatemerisation
approach using PCR amplified minor satellite monomers containing a binding site for
the cenpB protein. Concatemers will be used in a cotransformation approach as used
by Harrington et al or will be cloned into pac vectors containing telomeres and
selectable markers.
Reference List
Ebersole,T.A., Ross,A., Clark,E., Mcgill,N., Schindelhauer,D., Cooke,H., and Grimes,B.
(2000). Mammalian artificial chromosome formation from circular alphoid input DNA does not
require telomere repeats. Hum. Mol. Genet. 9, 1623-1631.
Ikeno,M., Grimes,B., Okazaki,T., Nakano,M., Saitoh,K., Hoshino,H., Mcgill,N.I., Cooke,H.,
and Masumoto,H. (1998). Construction of YAC-based mammalian artificial chromosomes.
Nature Biotechnology 16, 431-439.