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ARTIFICIAL CHROMOSOMES

E. Artificial Maize minichromosomes

Shawn R. Carlson, Gary W. Rudgers, Helge Zieler, Jennifer M. Mach, Song Luo, Eric Grunden, Cheryl Krol, Gregory P. Copenhaver, Daphne Preuss (2007) Meiotic Transmission of an In VitroAssembled Autonomous Maize Minichromosome PLoS Genet 3(10): e179. doi:10.1371/journal.pgen.0030179.

In constructing artificial chromosomes, one does not always have the luxury of having well defined centromeres, replication origins and telomeres available for use. One approach has been to delete most of a chromosome, and call the remaining part a minichromosome. The other approach is to try to find short sequences that give a functional chromosome when incorporated into a plasmid vector.

While large sections of the maize genome have been sequenced, including centromeric regions, the precise requirements for a centromere have not yet been defined. Thus, the strategy employed here was to  try to identify small fragments taken from centromeric regions that could give stable inheritance to a plasmid vector.

A note on replication origins: It is worth pointing out that for both mammalian and plant artificial chromosomes, animal and plant replication origins were not specifically included in the constructs. Rather it seems as if centromeric regions usually contain at least one replication origin, so if you get a functional centromere, you will probably get an origin of replication as well.

A note on telomeres: The maize minichromosomes described in this paper do not have telomeres, but rather are circular. Circular chromosomes replicate normally in eukaryotes. However, if sister chromatid exchange occurs (ie. recombination), the two mitotic products will end up as interlocking rings, which will then break when pulled in opposite directions during anaphase. This is seldom a problem for very small chromosomes, but are probably one of the reasons that large eukaryotic chromosomes need to be linear. See Lecture 19, Part 4 Ring Chromosomes.

1. Screen a BAC library for clones containing repeated sequences known to be associated with centromeres.

BAC vectors are plasmids designed to allow cloning of very large DNA fragments eg. > 50 kb to as high as 800 kb. Clones from a maize BAC library were hybridized with probes made from repeated sequences found in maize centromeric regions. Positive clones were selected for minichromosome construction.

2. Clone inserts with centromeric repeats into a vector containing a selectible marker and reporter gene.

BAC DNA was extracted from clones hybridizing with centromeric repeats. Putative centromeric sequences flanked by lox sites from the BAC vector were recombined in-vitro using Cre recombinase1 with pCHR758, to give circular Maize Mini Chromosomes.

MMCs each contain a centromeric insert, an nptII gene for resistance to the antibiotic neomycin (kanamycin), and a coding sequence for the red fluorescent protein DsRed. The DsRed gene is fused to a promoter and N-terminal coding sequence for proteasomal ubiquitin, which targets the chimeric protein to the nuclear proteasomal complexes. Thus, DsRed flourescence will be detected in nuclei of MMC cells.

Gene expression cassettes, which could potentially be used for expression of novel genes, are indicated by gray arrows. BglII restriction sites are indicated by triangles.

By crossing in the centromeric sequence, we can turn any plasmid that contains a lox site into a minichromosome.

from Shawn et al. doi:10.1371/journal.pgen.0030179
1Cre recombinase from bacteriophage P1 mediates recombination between 34 bp sequences known as  lox sites in vitro or in vivo.
Dale, E.C. and Ow, D.W. (1991) Gene transfer with subsequent removal of the selection gene from the host genome. Proc. Natl. Acad. Sci. USA 88:10558-10562. 

3. Transform maize tissue with constructs, and select for transformants on antibiotic media.

Recombinant MMC constructs were transformed into embryogenic maize tissue in culture were delivered into plant cells by biolistics. Briefly biolistic transformation is done by dipping gold microbeads in DNA solution, and then shooting the DNA-coated beads into plant cells using a "gene gun" which uses compressed gas to shoot particles at tissue. DNA dissolves off the beads, and is packaged into chromatin by spontaneous interaction of foreign DNA with histone proteins. Transformation in plants is often done using a handheld gene gun.

To select for transformants, tissue is propagated on media containing kanamycin.

4. Use FISH to screen transformed plantlets  for constructs maintained as independent chromosomes, rather than constructs incorporated into host chromosomes.

There are two possible ways to maintain the MMC constructs in plant cells. The construct could be propagated as minichromosomes, but it might also become integrated into host chromosomes. To distinguish between these possibilities, the authors used FISH.

Metaphase chromosomes from transformed plants were visualized by FISH with probes for centromeric repeats (red) and probes for the DsRed/nptII casette (green) and counterstained with DAPI dye, which stains DNA (blue). A shows a cell which contains a minichromosome, indicated by the arrow. A close up of the region with the minichromosome is shown in the inset, and in panels B - D. B - DAPI staining; C - DsRed/nptII probe; D - centromeric probe.


from Shawn et al. doi:10.1371/journal.pgen.0030179

Constructs incorporated into native chromosomes.

I - pCHR758 control vector with no centromeric repeats.

J - MMC integrated into host chromsomes

from Shawn et al. doi:10.1371/journal.pgen.0030179

5. Check for Mendelian inheritance of minichromosomes in subsequent generations.

One transformed line with a single copy of the MMC chromosome was designated as MMC1. The initial transformant generation, T0, was selfed, and fluorescence microscopy used to verify the transmission of MMC through two subsequent generations, T1 and T2.

from Shawn et al. doi:10.1371/journal.pgen.0030179
Visualization of MMC by DsRed fluorescence in T2 transgenic plants (ie. second generation progeny of the initial transformants, the T0 generation. A - closeup of maize leaf cells. B - bright field image of leaf tissue. C - fluorescent microscopy of the same leaf section as in B.

To qualify as a true chromosome, the MMC must be demonstrated to segregate according to Mendel's laws.

T0 plants were crossed with untransformed plants. Since each T0 would only be expected to have gotten a single copy of the minichromosome, rather than two, the T0 generation would be considered to be hemizygous for the MMC. Thus, crossing with untransformed plants should show a 1:1 segregation for the MMC. Selfing of T1 transformants should result in a 3:1 ratio of MMC versus null plants.

In several independent transformants, MMC did segregate according to these ratios. However, some lines showed a reduction in M_ progeny, suggesting that in those transformant lines, the MMC was unstable, and would be lost during meiosis.





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previous  page PLNT3140 Introductory Cytogenetics
Lecture 13, part 2 of 2
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