3. Flow Of
Seminar
23-Jan-16
Department of Genetics and Plant Breeding
3
Introduction.
Need of Synthetic Chromosomes.
Requirements for Synthetic
chromosomes.
Methods to develop Synthetic
chromosomes
Advantages and limitations.
Application of Synthetic chromosome
technology in crops- Case studies.
Future Aspects.
Conclusion.
4. Food In Fifty Years…??
4Department of Genetics and Plant Breeding23-Jan-16
5. Why make Synthetic chromosomes…???
5Department of Genetics and Plant Breeding23-Jan-16
8. • Gene stacking- difficult
• Transgene position
effects
First Generation Genetic Engineering
Second Generation Genetic Engineering
Synthetic Chromosomes
Delivery of large DNA sequences
Complete metabolic pathways
Genetic changes 100-kb to megabases (Mb)
Need.....???
8Department of Genetics and Plant Breeding23-Jan-16
9. Synthetic chromosomes are chromosome based
non-integrating vector system that is transmissible and
suitable for transfer of large genes, gene complexes
and/or multiple gene together with regulatory element
for safe, controlled and persistent gene expression
9
Department of Genetics and Plant Breeding23-Jan-16
10. 23-01-2016 DEPARTMENT OF PLANT BIOTECHNOLOGY 10
Demonstrated the successful
use of telomere truncation
in maize plants to produce
minichromosomes (2006)
created a synthetic
chromosome from yeast
paving the way for a new
field of study(2014)
Pillars of this new technology
James A Birchler
J. Craig Venter
11. “This has been sought for a long time in the plant world, and it should
open a number of new avenues. If we can do this in plants, a number of
advances could be done in agriculture that would not otherwise be
possible, from improved crops to inexpensive pharmaceutical production
to other applications in biotechnology”
James Birchler
MU College of Arts and Science
11Department of Genetics and Plant Breeding23-Jan-16
12. Requirements for Synthetic Plant
Chromosome
Basic requirements for Synthetic Plant Chromosome
1.Centromere
2. Telomere
3. Sufficient Chromatin
4. Selectable Marker transgene
5.Site specific recombination system
12Department of Genetics and Plant Breeding23-Jan-16
13. Centromere Components In Plants
Centromere – Requisite component of SC
Contains tandem repeated sequences of various
sizes, ~1 to 3 Mb.
CENH3,the Histone H3 varient of plants typical
of active centromeres, associates with this repeat
region.
Retrotransposons (Maize-CRM elements)
13Department of Genetics and Plant Breeding23-Jan-16
14. Telomere Components In Plants
Specialized structure which cap the ends of
eukaryotic chromosome.
Consist of highly conserved long array of short
tandemly repeated sequences.
e.g. TTTAGGG in A. thaliana
TTAGGG in Homo sapiens
TTAGG in insects
Average length: 3-40 kb
(Burr et al., 1992)
Functions: 1. Maintaining the structural integrity
2.Ensure complete replication of extreme ends of
chromosome
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15. Methods To Develop Synthetic
Chromosome
15Department of Genetics and Plant Breeding23-Jan-16
16. Bottom up method
• de novo assembly of cloned chromosomal
components, such as
centromeric and telomeric sequences,
a selective marker gene and
genomic DNA that contains a replication origin.
This method is well established in
Yeast (Murray and Szostak, 1983)
Mammalian cells (Harrington et al.,1997)
16Department of Genetics and Plant Breeding23-Jan-16
17. Reasons for failure
• Construction yeast artificial chromosome is used as a model for
bottom-up method.
• Centromere specification in yeast is unusual among eukaryotes
and Centromeres in higher eukaryotes have diffuse organization
(Henicoff et al., 2001).
e.g. Centromeric repeats of barley (Hordeum vulgare) have been
shown to be neither necessary nor sufficient to establish a functional
centromeric activity (Nasuda et al., 2005) .
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18. Epigenetic Aspects Of Centromere Specification
• Mechanism by which centromeres are established, maintained,
and function remain a mystery.
• A chromosome was found that did not contain detectable
centromere repeats but till organized a kinetochore at a specific
site that was associated with centromere activity in barley
(Nasuda et al.,2005).
• Substitution of the histone H3 by CENH3(CENP-A) in
centromeric nucleosomes is crucial for kinetochore formation.
18Department of Genetics and Plant Breeding23-Jan-16
19. So what is the solution…???
23-Jan-16 Department of Genetics and Plant Breeding 19
20. Top down method
Based on chromosome fragmentation or
truncation.
This method utilizes the insertion of
telomere sequences into existing
chromosomes.
This sequence signals for new telomeres
which causes the truncation of the
chromosome.
insertion of new genes for desired traits.
20Department of Genetics and Plant Breeding23-Jan-16
21. Telomere-mediated Truncation
Aim- To whittle away the chromosome arms using
transformation of telomere repeats.
It bypasses the complications of the epigenetic aspects of
centromere specification.
It works robustly in plants & can be used to produce engineered
minichromosomes with endogenous centromeres.
Construct- Genes of interest, Site-specific recombination
cassettes, Telomere repeats.
(Yu et al., 2006)
21Department of Genetics and Plant Breeding23-Jan-16
22. 23-Jan-16 Department of Genetics and Plant Breeding 22
Adding of genes to a engineered minichromosome
23. This approach of telomere mediated
chromosome truncation was first shown by
Farr et al.,1991 i.e. introduction of cloned
telomeric repeats into cultivated cells may
truncate the distal portions of chromosomes
by the formation of new telomeres at
integration sites in mammalian cell lines.
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24. Type of
truncation
Description Pros Cons
Truncation of B
chromosomes
In plant species that
harbour Bs,
truncation of B
chromosome arms
can be selected.
Loss of chromosome
segments from Bs
does not affect the
viability of the plant.
Bs are not present in
all plant species. This
approach is applicable
only to those species
that have or will
accept Bs.
Truncation of A
chromosomes in
tetraploid
background
Truncation of A
chromosome can be
rescued
in a tetraploid
background.
Tetraploid plants can
tolerate truncation of
chromosome arms.
This approach will
work readily in
natural polyploids.
Tetraploids would
need to be generated
in diploid species.
(Gaeta et al.,2012)
24Department of Genetics and Plant Breeding23-Jan-16
25. 25Department of Genetics and Plant Breeding23-Jan-16
A chromosome truncation B chromosome truncation
Particle bombardment
Truncating plasmid
26. Telomere truncation of different types of
chromosomes
(a) A chromosome
(b) B chromosome
(c) Telocentric chromosome
26Department of Genetics and Plant Breeding23-Jan-16
28. Minichromosomes
• extremely small version of a chromosome.
• By minimizing the amount of unnecessary genetic information
on the chromosome and including the basic components
necessary for replication,we can construct this chromosomal
plotform,so that we can insert new genes.
28Department of Genetics and Plant Breeding23-Jan-16
29. B Chromosome Based Minichromosomes
B chromosome based minichromosome is interesting-
Reasons:-
1. Naturally occurring supernumerary chromosome
2. Basically inert, small size – no phenotype
(Jones et al., 2003)
3. No developmental & transmission problem
4. Easy detection of B chromosome derivatives
(Kato et al., 2005)
5. No report of recombination with A chromosome set
Minimal detrimental effect on host genome
29Department of Genetics and Plant Breeding23-Jan-16
30. 23-Jan-16 Department of Genetics and Plant Breeding 30
Generalized scheme for the production of engineered
minichromosomes in maize
31. J. A. Birchler et al.,2010
31
Department of Genetics and Plant Breeding23-Jan-16
Maintenance Of mini B chromosomes in the population
33. • Fecilitates in understanding fundamental questions
about chromosomal structure and function.
• Synthetic chromosomes in plants are likely to have
more applications than in other taxa.
• This approach for construction of engineered
chromosomes can be easily extended to other plant
species
• allows for the stacking of genes side-by-side on the
same chromosome thus reducing likelihood of
segregation of novel traits.
Advantages
33Department of Genetics and Plant Breeding23-Jan-16
34. • Functional genomic studies could use
minichromosomes as a platform for adding
specific genes
• Next generation vectors for human gene
therapy & plant genetic engineering
• stable expression & maintenance of
multiple transgenes in one genome.
• Mass production of foreign proteins,
pharmaceuticals, or useful metabolites.
23-Jan-16 Department of Genetics and Plant Breeding 34
35. Limitations
• Small chromosomes do not always pair
homologously in meiotic prophase.
• low meiotic transmission rate.
• pollen abortion.
• Regeneration of plants is difficult.
23-Jan-16 Department of Genetics and Plant Breeding 35
38. • In this report, they demonstrated that 2.6 kb of Arabidopsis
telomeric repeats were efficient for telomere-mediated
chromosomal truncation in maize.
• They also showed that internally integrated telomeric
sequences are stable in the genome.
23-Jan-16 Department of Genetics and Plant Breeding 38
39. 23-Jan-16 Department of Genetics and Plant Breeding 39
Constructs Transgenic
plants
Transgenic loci
pWY76 93 123(57 at distal loci)
pWY86 83 108 (61 at distal loci)
pWY96 44 58 (11 at distal loci)
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Cytological detection of chromosomal truncations
pWY86 pWY76
pWY86 pWY86
B77
T87
B37
B44
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Restriction mapping of the positions of
transgenes by a Southern blot.
42. Summary
• 2.6-kb direct repeat of Arabidopsis telomeric
sequence was used in two constructs, pWY76 and
pWY86, to test the ability of telomeric sequences to
cause chromosomal truncations.
• Direct evidence for chromosomal truncation came
from the results of FISH karyotyping, which revealed
broken chromosomes with transgene signals at the
ends.
• This technology will be useful for chromosomal
engineering in maize as well as other plant species.
23-Jan-16 Department of Genetics and Plant Breeding 42
44. EXPERIMENTAL PROCEDURES
Plant material:
Embryogenic calli derived from Immature embryos of rice
telotrisomic line with Telo-12L derived from indica rice cultivar
Zhongxian 3037.
Callus induction:
• Immature embryos were cultured on N6D2 medium at 28 ͦC in
the dark for callus induction.
• Embryogenic calli were subcultured every 2–3 weeks and used
for transformation by bombardment.
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46. Gene transformation:
• 1.5 mg 0.6 μM gold particles were coated with 2 μg plasmid mixture
and bombarded to rice calli with a PDS 1000/He particle delivery
system.
• After 16 hours,the calli were transferred onto N6D2 medium
supplemented with 25 mg/L hygromycin and cultured at 28 ͦC in the
dark for 2 weeks.
• and then selected by subculture on N6D2 supplemented with 50 mg/L
hygromycin every 3 weeks until the resistant calli emerged.
• The resistant clones were maintained on N6D2 medium supplemented
with 25 mg/L hygromycin at 28 ͦC by subculture every 3 weeks.
Chromosome preparation from callus
• Fast growing calli on selection medium after 7 days subculture were
selected to check chromosomal truncation and mini-chromosome
formation in transgenic rice using FISH.
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47. (b)Transgenic clone with one distal signal
47
(a)Transgenic clone with one internal signal
Fluorescence in situ hybridization(FISH) detection of transgenes
48. Rice transformation and transgene distributions
No. of
bombarded
calli
No. of
resistance
clones
No. of
Transgenic
events
checked
No. of
Transgene
signals
No of
internal
transgenes
No. of
distal
transgenes
Ratio (%)
of distal
transgenes
Control (a) 519 44 19 32 31 1 3.1
4–6-month-
old calli (b)
4512 536 122 178 121 57 32.0
18-month-
old calli (c)
854 111 94 138 84 54 39.1
Total no.
(b + c)
5366 647 216 316 205 111 35.1
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The control was bombarded with only pCAMBIA1301
49. 23-01-2016 DEPARTMENT OF PLANT BIOTECHNOLOGY 49
(a–c) Mini-chromosomes
with one transgene signal:
(a) Clone 1008-100
(b) Clone 1004-111
(c) Clone 1004-015.
(d) Clone 1004-011, mini-
chromosome with
transgene signals at both
ends of the chromosome.
Rice mini-chromosomes from
telomere truncation.
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Clone 1004-111
• One chromosome 12 and one Telo-
12L are shown in this clone.
• The mini-chromosome
(arrowhead) is probably originated
from chromosome 12 truncation
Origin of mini-chromosomes
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Clone 1008-100
• Three chromosomes 12L are identified
• Original Telo-12L was not found.
• The missing 12L is probably truncated to
produce the mini-chromosome
(arrowhead).
• Chromosome 12 is probably duplicated
to produce an extra chromosomes 12.
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Distal transgene in
control without
telomere sequence
Distal transgene
with telomere
sequence
Maize
chromosomal
truncation
19.0 % 46.3-56.5 %
Rice chromosomal
truncation 3.1 % 35.1 %
These results were compared with previous report of
maize chromosomal truncations by Yu et al.,2006
53. Summary
• Telotrisomic rice line was used for mini-chromosome construction.
• minichromosomes were recovered from transgenic clones by monitoring
hygromycin-resistant calli with FISH.
• All these mini-chromosomes were maintained stably in cell cultures for
over 2 years.
• The construction of mini-chromosomes in rice will provide a platform for
future artificial chromosome-based genetic engineering of this plant for
multiple gene expressions.
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54. • develop a mini B chromosome-
based genomic cloning system.
• combination with haploid
breeding.
• Genome editing techniques.
• Deliver genes that benefit the
agricultural, nutritional, energy and
pharmaceuticals sectors.
23-Jan-16 Department of Genetics and Plant Breeding 54
unlike de novo artificial chromosome construction, which requires cloning of large centromere sequences from the plant species for which artificial chromosomes are to be constructed. It has been argued that with one telomere truncation construct, almost all transformable plant species can be engineered by chromosomal truncation (Yu et al., 2006) because the telomere sequence is conserved in most plant species except some genera in the Asparagales
that could be utilized as a platform for inserting genes into the plant genome
Centromeres are essential for chromosome inheritance and genome stability. Centromeric proteins, including the centromeric histone centromere protein A (CENP-A), define the site of centromeric chromatin and kinetochore assembly. In many organisms, centromeres are located in or near regions of repetitive DNA. However, some atypical centromeres spontaneously form on unique sequences. These neocentromeres, or new centromeres, were first identified in humans, but have since been described in other organisms. Neocentromeres are functionally and structurally similar to endogenous centromeres, but lack the added complication of underlying repetitive sequences. Here, we discuss recent studies in chicken and fungal systems where genomic engineering can promote neocentromere formation. These studies reveal key genomic and epigenetic factors that support de novo centromere formation in eukaryotes.
Adding genes to an engineered minichromosome. (A) An initial minichromosome is depicted with an endogenous centromere (circle), a site for addition of new sequences (XXX), and the original transgenes (genes 1–3) added at the time of truncation and capped by telomere sequences (in the color teal). (B) Additional genes could be added to a minichromosome by site-specific recombination (denoted by X) to the target site (XXX) by the introduction of a circular molecule in the presence of the appropriate recombinase. Recombination will place new genes (genes 4–6) onto the minichromosome. The stacking system described by Ow [20] or modification thereof will allow continued additions to grow the chromosome.
Truncation of A and B chromosomes. Introduction of the truncating plasmid with the selectable marker for herbicide resistance and the telomere array into cells carrying B chromosomes will result in truncated A chromosomes in some cases or truncated B chromosomes. The yellow circle represents a petri dish with young maize embryos for transformation via particle bombardment. The orientation of the truncating plasmid will determine whether the truncation can be selected. If the truncating plasmid is attached to an acentric fragment it will be lost. If the truncating plasmid is oriented so that it is attached to a centromere, then the truncation event can be selected and will contain the genes carried on the plasmid at the end of the chromosome.
(a) A normal chromosome has two chromosome arms (blue) and a centromere (green). Two chromosome arms need to be truncated by incoming telomere repeats (red) to make a mini-chromosome.
(b) B chromosome has a very small short arm (blue), and mini-chromosome can be produced by a truncation of the long arm (blue).
(c) Telocentric chromosome has only one arm (blue), and mini-chromosome can be produced by only one chromosomal truncation.
The integration of transgenes containing telomere repeats can be resolved in two ways. If both ends of the transgene are repaired and ligated into the location of a break, then transgene integration occurs (bottom right). Alternatively, if the transgene terminus containing telomere repeats is recognized by telomere-binding proteins,leading to telomere extension and capping, and the other end of the transgene is ligated, then the result is a truncated centromere-containing chromosome with a terminal transgene locus and an acentric fragment (top right).
Telomere-mediated truncation of maize chromosomes can be induced following transformation with a transgene cassette that contains
telomere repeats by (a) particle bombardment or (b) Agrobacterium-mediated transformation. Transgenes introduced by either method
insert at the site of a DNA break, and this integration involves DNA repair mechanisms. The locations of DNA breaks in the genome
are thought to be random.
Increasing the copy number of truncated mini B chromosomes. By introducing a normal B chromosome into the genotype, truncated B chromosomes can undergo nondisjunction at the second pollen mitosis. Using this property, the truncated B chromosomes can be accumulated in subsequent generations by screening and selecting plants with higher numbers of truncations. Green arrows denote B truncations. Red arrows denote normal B chromosomes.
because it does not rely on cloned centromere sequences, which are species-specific.
Coping with the meiotic transmission of small chromosomes is an area for future development in the field. Small chromosomes often lack sister chromatid cohesion in meiosis I and thus the chromatids separate at this division whereas normal sized chromosomes do not. This fact precludes a 100% transmission if a pair of minichromosomes is present because there will be microspores at the end of meiosis that will be missing a minichromosome because of sister separation in meiosis I and random distribution in meiosis II. Also small chromosomes often cannot find their pairing partners in meiosis, which is a fact that also militates against complete transmission from a pair of minichromosomes because they independently assort rather than segregate from each other.
The three constructs were used to transform maize immature embryos by an Agrobacterium-mediated gene transformation method.In this report, a 2.6-kb direct repeat of Arabidopsis telomeric sequence was used in two constructs, pWY76 and pWY86, to test the ability of telomeric
sequences to cause chromosomal truncations.and they included a HPT gene-expression cassette(selectable marker) in pWY76, pWY96,
which contains the elements common to both pWY76 and pWY86 but lacks the 2.6-kb telomere sequence (Fig. 1) was used as a control to test the function of telomeric sequences and was used as a FISH probe to detect all transgenes from pWY76, pWY86, and pWY96 transformations.
To localize transgenes and to detect chromosomal truncations, metaphase chromosomes from T0 transgenic plant root tips were probed with a pWY96 probe plus the karyotyping mixture.
B37Metaphase chromosomes were hybridized with pWY96 probe (red). Arrows denote the transgene truncation sites (white arrows)
and the corresponding sites on the homologues (gray arrows). (A) pWY86 transgenic event B77 with a chromosome 3 short-arm truncation. (Inset) The chromosome 3 pair with (Left) and without (Right) the transgene (red). (B) pWY76 transgenic event T87 with a chromosome 1 short-arm terminal-knob truncation. (Inset) Chromosome 1 homologues with (Upper) and without (Lower) the transgene (red). (C and D) pWY86 transgenic events B37 and B44 with truncations of chromosome 4 long-arm terminal subtelomeric 4–12-1 sequence (green). (Insets) Chromosome 4 homologues with (Upper) and without (Lower) the transgene (red). Chromosome 4 can be identified by the Cent4 hybridization (white) at their centromeres.
In all these cases, the deletion of a chromosomal segment was correlated with the integration of the transgene at the corresponding terminal position. However, many truncations might not be revealed by this method. For example, small deletions caused by truncation might not be observed on highly condensed somatic chromosomes.Because of the limitations of the cytological analysis, a Southern hybridization method was performed on selected transgenic
Chromosomal truncations result in synthesis of new telomeres at broken ends to maintain chromosome stability. The seeded telomeres are heterogeneous in size because the telomerase adds different numbers of telomeric repeats to the chromosomal termini in each cell. A Southern hybridization will reveal the heterogeneous telomeres as a smear if DNA is digested with a restriction enzyme that cleaves a nearby internal site.
Smear patterns were observed for two events (B37 and B77) that were characterized by FISH and that had obvious truncations
These results demonstrate that telomere-mediated chromosomal truncation operates in plant species.
This technology will be useful for chromosomal engineering in maize as well as other plant species.
the unique DNA recombination event that took place in one plant cell, which was then used to generate entire transgenic plants
Transgene can be detected in 4 minichromosomes with the pCAMBIA 1301 probe.3d. One mini-chromosome was shown to have two transgenes that flank a centromere (Figure 3d), that resulted probably from chromosomal truncations in both of the arms.
Three clones were checked with chromosome arm specific probes to find out the origins of mini chromosomes.These clones were all created from a telotrisomic line that contained one 12L telocentric chromosome.two chromosomes 12 and 12L would be present if there is no chromosomal truncation.
The construction of mini-chromosomes in rice, an economically important crop, will provide a platform for future
artificial chromosome-based genetic engineering of rice for stacking multiple genes.
Recent findings suggest that minichromosomes can be combined with haploid breeding to facilitate transgene transfer to many new varieties or to assess how a transgene behaves in a different genetic background. Using this procedure, it would then be possible to incorporate the minichromosome into new inbred lines much more quickly than by repeated backcrossing. Also, the mere ability to place engineered minichromosomes into haploids would allow the evaluation of the interaction of the included transgenes with various established or novel genomic constitutions. Site-specific recombination systems & zinc finger nucleases are valuable tools for marker gene removal & gene targeting. Such technologies could be applied to AC
As the population of world goes on incresing. There is a need to find new techniques to improve the food production. Crop production strategies can eemploy the techniques from many deciplines.. One of such decipline is transgenic approach.
