HYBRIDIZATION & HAPLOID PRODUCTION
Introduction
WIDE HYBRIDIZATION
INTER-SPECIFIC HYBRIDIZATION
Barriers to distant hybridization
Techniques to overcome barriers
Haploids and Doubled Haploids in Plant
Production of haploids and doubled haploids
a) Induction of maternal haploids
Wide hybridization
3. In vitro induction of maternal haploids – gynogenesis
Induction of paternal haploids – Androgenesis
Production of Homozygous Diploid Plants
Application of Haploids in Plant Breeding
Importance and Implications of Anther and Pollen Culture
Invitro culture of unpollinated ovaries and ovules represents an alternative for the production of haploid plant
First successful report on the induction of gynogenic haploid was in barley by San Noeum in 1976
Haploid plants are obtained from ovary and ovule culture of rice, wheat, maize, sunflower, tobacco, poplar, mulberry etc
Whites or MS or N6 inorganic salt medium supplement with growth substances are used
HYBRIDIZATION & HAPLOID PRODUCTION
Introduction
WIDE HYBRIDIZATION
INTER-SPECIFIC HYBRIDIZATION
Barriers to distant hybridization
Techniques to overcome barriers
Haploids and Doubled Haploids in Plant
Production of haploids and doubled haploids
a) Induction of maternal haploids
Wide hybridization
3. In vitro induction of maternal haploids – gynogenesis
Induction of paternal haploids – Androgenesis
Production of Homozygous Diploid Plants
Application of Haploids in Plant Breeding
Importance and Implications of Anther and Pollen Culture
Invitro culture of unpollinated ovaries and ovules represents an alternative for the production of haploid plant
First successful report on the induction of gynogenic haploid was in barley by San Noeum in 1976
Haploid plants are obtained from ovary and ovule culture of rice, wheat, maize, sunflower, tobacco, poplar, mulberry etc
Whites or MS or N6 inorganic salt medium supplement with growth substances are used
Haploid Production - Techniques, Application & Problem ANUGYA JAISWAL
Haploid is applied to any plant originating from a sporophyte (2n) and containing (n) number of chromosomes.
Artificial production of haploids was attempted through distant hybridization, delayed pollination, application of irradiated pollen, hormone treatment and temperature shock.
The artificial production of haploids until 1964 was attempted through:
1. Distant hybridization
2. Delayed pollination
3. Application of irradiated pollen
4. Hormone treatments
5. Temperature shocks
The development of numerous pollen plantlets in anther cultures of Datura innoxia, first reported by two Indian scientists (Guha and Maheshwari, 1964, 1966), was a major breakthrough in haploid breeding of higher plants.
The technique of haploid production through anther culture ('anther - androgenesis') has been extended successfully to numerous plant species, including many economically important plants, such as cereals and vegetable, oil and tree crops.
Organogenesis, in plant tissue cultureKAUSHAL SAHU
Introduction
Definition
Types of organogenesis
Organogenesis through callus formation (indirect organogenesis)
Growth regulators for indirect organogenesis
Organogenesis through adventitious organ (direct organogenesis)
Growth regulators for direct organogenesis
Factor affecting the soot bud differentiation
Organogenic differentiation
Application of organogenesis
Conclusion
References
INTRODUCTION
2. HISTORY
3. BASIC COMPONENT OF MEDIA
1. Inorganic nutrient
2. organic supplements
3. Carbon and energy source
4. Growth Regulators
5. Solidifying Agent
6. PH
4. TYPES OF MEDIA
5. MS MEDIA
6. IMPORTANCE
7. CONCLUSION
8. REFERANCE
☺INTRODUCTION
☺Bt COTTON
☺MAJOR PESTS OF COTTON
☺MODE OF ACTION OF Bt GENE
☺ADVANTAGES
☺DISADVANTAGES
☺CONCLUSION
☺REFERENCES
Genetically modified variety of cotton that produces an insecticide whose gene has been derived from a soil bacterium called Bacillus thuringiensis (Bt).
Three types of toxins.
A total of 229 cry toxins ( cry1Aa to Cry72Aa), cyt toxins ( cyt 11Aa to cyt3Aa) and 102 vip toxins( vip1Aa1 to vip4Aa1) have been discovered.
Haploid Production - Techniques, Application & Problem ANUGYA JAISWAL
Haploid is applied to any plant originating from a sporophyte (2n) and containing (n) number of chromosomes.
Artificial production of haploids was attempted through distant hybridization, delayed pollination, application of irradiated pollen, hormone treatment and temperature shock.
The artificial production of haploids until 1964 was attempted through:
1. Distant hybridization
2. Delayed pollination
3. Application of irradiated pollen
4. Hormone treatments
5. Temperature shocks
The development of numerous pollen plantlets in anther cultures of Datura innoxia, first reported by two Indian scientists (Guha and Maheshwari, 1964, 1966), was a major breakthrough in haploid breeding of higher plants.
The technique of haploid production through anther culture ('anther - androgenesis') has been extended successfully to numerous plant species, including many economically important plants, such as cereals and vegetable, oil and tree crops.
Organogenesis, in plant tissue cultureKAUSHAL SAHU
Introduction
Definition
Types of organogenesis
Organogenesis through callus formation (indirect organogenesis)
Growth regulators for indirect organogenesis
Organogenesis through adventitious organ (direct organogenesis)
Growth regulators for direct organogenesis
Factor affecting the soot bud differentiation
Organogenic differentiation
Application of organogenesis
Conclusion
References
INTRODUCTION
2. HISTORY
3. BASIC COMPONENT OF MEDIA
1. Inorganic nutrient
2. organic supplements
3. Carbon and energy source
4. Growth Regulators
5. Solidifying Agent
6. PH
4. TYPES OF MEDIA
5. MS MEDIA
6. IMPORTANCE
7. CONCLUSION
8. REFERANCE
☺INTRODUCTION
☺Bt COTTON
☺MAJOR PESTS OF COTTON
☺MODE OF ACTION OF Bt GENE
☺ADVANTAGES
☺DISADVANTAGES
☺CONCLUSION
☺REFERENCES
Genetically modified variety of cotton that produces an insecticide whose gene has been derived from a soil bacterium called Bacillus thuringiensis (Bt).
Three types of toxins.
A total of 229 cry toxins ( cry1Aa to Cry72Aa), cyt toxins ( cyt 11Aa to cyt3Aa) and 102 vip toxins( vip1Aa1 to vip4Aa1) have been discovered.
Plant Chromosomes: European Cytogeneticists outline: Trude Schwarzacher and P...Pat (JS) Heslop-Harrison
An overview of plant molecular cytogenetics. The lecture Trude Schwarzacher presented to the ECA conference Strasbourg in July 2015 is http://www.slideshare.net/PatHeslopHarrison/trude-schwarzacher
Molecular Systematics provides a solid conceptual basis for the evolutionary history of organisms. Molecular systematics is the study of DNA and RNA sequences to infer evolutionary links across organisms. Molecular approaches/ techniques provide excellent resources for the study of evolution and phylogeny.
A plant genome project aims to discover all genes and their function in a particular plant species.
The main objective of genomic research in any species is to sequence the whole genome and functions of all the different coding and non-coding sequences.
These techniques helped in preparation of molecular maps of many plant genomes.
Plant genome projects initially focused on a few model organisms that are characterized by small genomes or their amenability to genetic studies
Since sequencing technologies have moved on, sequencing cost have dropped and bioinformatics tools advanced, the genomes of many plant species including the enormous genome of bread wheat have been assembled
Genome sequencing projects have been carried out on all three plant genomes: the nuclear, chloroplast and mitochondrial genomes
This opened venues for advanced molecular breeding and manipulation of plant species, but also have accelerated phylogenetics studies amongst species
Several excellent curated plant genome databases, besides the general nucleotide data base archives, allow public access of plant genomes
Gene Editing: An Essential Tool For Plant BreedingNoreen Fatima
Gene editing to remove the specie barriers. In many crops it have been used for improvement of crops for particular attribute. in this many tools have been used . these tools consist of nucleases that cut the DNA. So that is used for gene regulation, gene knock out and also use for point mutations.
Los días 7 y 8 de mayo organizamos en la Fundación Ramón Areces con la Fundación General CSIC el Simposio Internacional 'Microbiología: transmisión'. La "transmisión" en microbiología hace referencia al proceso por el que material genético es transferido de una célula a otra, de una población a otra. Es un proceso clave para entender el origen y la evolución de los seres vivos. El objetivo de esta reunión era conocer mejor la logística de la transmisión para ser capaces de modular o suprimir algunos procesos de transmisión dañinos.
Similar to SYNTHETIC CHROMOSOME PLATFORMs IN PLANTS: CONCEPTS & APPLICATIONs (20)
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
I will finally argue that deep variability is both the problem and solution of frictionless reproducibility, calling the software science community to develop new methods and tools to manage variability and foster reproducibility in software systems.
Exposé invité Journées Nationales du GDR GPL 2024
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Toxic effects of heavy metals : Lead and Arsenicsanjana502982
Heavy metals are naturally occuring metallic chemical elements that have relatively high density, and are toxic at even low concentrations. All toxic metals are termed as heavy metals irrespective of their atomic mass and density, eg. arsenic, lead, mercury, cadmium, thallium, chromium, etc.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
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Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
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In this book, we use conservation of energy techniques on a fluid element to derive the Modified Bernoulli equation of flow with viscous or friction effects. We derive the general equation of flow/ velocity and then from this we derive the Pouiselle flow equation, the transition flow equation and the turbulent flow equation. In the situations where there are no viscous effects , the equation reduces to the Bernoulli equation. From experimental results, we are able to include other terms in the Bernoulli equation. We also look at cases where pressure gradients exist. We use the Modified Bernoulli equation to derive equations of flow rate for pipes of different cross sectional areas connected together. We also extend our techniques of energy conservation to a sphere falling in a viscous medium under the effect of gravity. We demonstrate Stokes equation of terminal velocity and turbulent flow equation. We look at a way of calculating the time taken for a body to fall in a viscous medium. We also look at the general equation of terminal velocity.
Phenomics assisted breeding in crop improvementIshaGoswami9
As the population is increasing and will reach about 9 billion upto 2050. Also due to climate change, it is difficult to meet the food requirement of such a large population. Facing the challenges presented by resource shortages, climate
change, and increasing global population, crop yield and quality need to be improved in a sustainable way over the coming decades. Genetic improvement by breeding is the best way to increase crop productivity. With the rapid progression of functional
genomics, an increasing number of crop genomes have been sequenced and dozens of genes influencing key agronomic traits have been identified. However, current genome sequence information has not been adequately exploited for understanding
the complex characteristics of multiple gene, owing to a lack of crop phenotypic data. Efficient, automatic, and accurate technologies and platforms that can capture phenotypic data that can
be linked to genomics information for crop improvement at all growth stages have become as important as genotyping. Thus,
high-throughput phenotyping has become the major bottleneck restricting crop breeding. Plant phenomics has been defined as the high-throughput, accurate acquisition and analysis of multi-dimensional phenotypes
during crop growing stages at the organism level, including the cell, tissue, organ, individual plant, plot, and field levels. With the rapid development of novel sensors, imaging technology,
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Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
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Salas, V. (2024) "John of St. Thomas (Poinsot) on the Science of Sacred Theol...Studia Poinsotiana
I Introduction
II Subalternation and Theology
III Theology and Dogmatic Declarations
IV The Mixed Principles of Theology
V Virtual Revelation: The Unity of Theology
VI Theology as a Natural Science
VII Theology’s Certitude
VIII Conclusion
Notes
Bibliography
All the contents are fully attributable to the author, Doctor Victor Salas. Should you wish to get this text republished, get in touch with the author or the editorial committee of the Studia Poinsotiana. Insofar as possible, we will be happy to broker your contact.
2. Why Synthetic/ Artificial Chromosome (AC)?
Methods to develop AC
Case study
Amendments in AC
Applications
Summary Points
Future Direction
Conclusion
OUTLINES OF THE SEMINAROUTLINES OF THE SEMINAR
3. Need.....???
Traditional genetic engineering
1) The transformation process
often results in the integration of foreign genes into an
endogenous gene and disrupts its function
2) Transgenes can be influenced by upstream or downstream
regulatory elements around them, that is, they are subject
to position effects under these circumstances
3) Only a single gene or a few genes can be expressed
4) Most importantly, Gene stacking or pyramiding
( Halpin, 2005)
4. First Generation Genetic Engineering
• Gene stacking- difficult
• Transgene position effects
• Insertion-site complexity
Second Generation Genetic Engineering
Synthetic Chromosome
Delivery of large DNA sequences
Complete metabolic pathways
Genetic changes 100-kb to megabases (Mb)
Goyal et al., 2009
Need.....???
5. Artificial Chromosome:
It refers to any nonintegrating vector that is transmissible and has the
ability to harbor large amounts of DNA
In bacteria – circular
In yeast, animals & plants- linear
First constructed in yeast and E. coli systems
(Clarke & Carbon, 1980)
Requirements for chromosomal maintenance and stability
- Centromeres
- Telomeres
- Origins of replication
(Murray & Szostak, 1983)
Introduction
7. Centromere Components In Plants
Centromere – Requisite component of AC
Contains tendem repeated sequences of various sizes, ~1 to 3 Mb
(Copenhaver, 2003)
In grass family, there is small (156 bp) repeat sequences
(Jin et al., 2004)
Study of mis-division of centromeres: The core functional B centromere is in the
range of 300 kb or larger in maize
(Jin et al., 2005)
Retrotransposons
(Neumann et al., 2009)
In maize, the tendem array size of centromere repeat C (CentC)- 700 kb to 2.7Mb
but the entire length is not necessarily associated with CENH3
(Wolfgruber et al., 2009)
8. Epigenetic Aspects Of Centromere Specification
For construction of AC, Yeast as a model- old concept
Centromere specification in yeast is unusual among
eukaryotes
Centromeres in higher eukaryotes have diffuse organization
(Henicoff et al., 2001)
Kinetochores form over apparently unique DNA, which is referred to as a
neocentromere
Repeats are not a necessary determinant of the sites of kinetochore formation
(Malik &Henikoff, 2009)
Mechanism by which centromeres are established, maintained, and function
remain a mystery
(Dalal, 2009)
9. 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 on an average
(Burr et al., 1992)
Functions: 1. Maintaining the structural integrity
2. Ensure complete replication of extreme ends of chromosome
Surovtseva et al., Mol. Cell, 2009
10. 1. Yeast Artificial Chromosome (YAC)
YAC is plasmid into which yeast genes have
been inserted
Developed by Murray & Szostak in 1983
Capacity- 3000 kb
YACs- Largest capacity vectors available
Advantage: Can be used to express
eukaryotic proteins that require post
translational modification
Disadvantages: 1. Less stable than BACs
2. Chimerism
Artificial Chromosome As Vectors
Murray and Szostak, Nature,
1983
11. 2. Bacterial Artificial Chromosome (BAC)
Construction is based on F-plasmid
Recombinants can be identified by Lac selection
Capacity- 350 kb
Uses: 1. Extensively used to sequence the genome of organisms
e.g.- HGP
2. Being utilized to a greater extent in modelling genetic diseases
e.g.- Alzheimer's disease, Cancer
3. Infectious viral clones
e.g.- herpesviruses, poxviruses
CONTD…CONTD…
Shizuya et al., Proc. Natl. Acad.
Sci., 1992
12. 3. Human Artificial Chromosome (HAC)
HAC is synthetically produced vector DNA, possessing the characteristics
of human chromosome
Developed by Harrington et al., in 1997
Size- 1/10th
to 1/5th
of human chromosome- 6 to 10 Mb
Mitotically stable for up to six months
Advantage: 1. Can carry human genes that are too long
2. Can be used for gene therapy
3. Useful in expression studies as gene transfer vectors
CONTD…CONTD…
Harrington et al., Nat. Genet. 1997
13.
14. Bottom-up Method
Building chromosomes by de novo assembling their component parts
This approach has been used for assembly of HAC
(Harrington et al., 1997)
Upon delivery, DNA is subjected to rearrangements resulting in AC larger than
the original construct
(Lim & Farr, 2004)
Not yet used in plant due to high complexity & limited understanding about plant
centromere organization
(Houben & Schubert, 2007)
15. Top-down Method
Based on chromosome fragmentation or truncation
Achieved by- 1. Irradiation
2. Telomere mediated truncation
Irradiation:
Example- An unstable maize minichromosome comprising part of the short arm of
chromosome 10 has been recovered as a result of pollen irradiation
(Brock and Pryor, 1996)
Disadvantage: Unstable
16. 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)
17. B Chromosome Based Minichromosomes
We can produce A & B chromosome based minichromosomes by this method
but 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
24. RESULTS...
Fig. 1. Chromosomal truncation constructs pWY76 and pWY86 and the control construct pWY96.
Tvsp- terminator from soybean vegetative storage protein gene; Bar- bialophos resistance gene as a selection marker
gene; P35S- 35S promoter from cauliflower mosaic virus; Tnos- Nos terminator from Agrobacterium; Tmas- Mas
terminator from Agrobacterium; Pnos- Nos promoter from Agrobacterium; Pmas1- Mas promoter from Agrobacterium;
lox and FRT, site-specific recombination sites; HPT- hygromycin B resistance gene; GFP- green fluorescent protein
gene; DsRed- red fluorescent protein gene; FLP, recombinase gene; Telomeres, telomere units of pAtT4 isolated from
Arabidopsis . Arrows designate the direction of transcription.
Development of Constructs
26. CONTD…
Fig. 2. Cytological detection of chromosomal truncations. Metaphase chromosomes were hybridized with pWY96 probe
(red) and mixtures of repetitive sequence probes, CentC (green), knob (green), subtelomere 4–12-1 clone (green), and
Cent4 (white). Arrows denote the transgene truncation sites (white arrows) and the corresponding sites on the homologues
(gray arrows). (A) pWY86 ransgenic 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. (Scale bar, 10 m.)
pWY86 B77 3S T pWY76 T87 1S T
pWY86 B37 4L T pWY86 B44 4L T
27. CONTD…
Fig. 4. Telomere sequences detected in internal chromosome locations.
Metaphase chromosomes of a pWY86 transgenic line were probed with a pWY96 probe (red) and a telomere
probe (green). Chromosomes were stained with DAPI. Arrowheads indicate the internal telomere (green) and
transgene (red) signals. Arrowheads in the enlarged images from the top to the bottom panels denote merged
transgene (red) and telomere (green) signals, and telomere only (green) and transgene only (red). (Scale bar,
10 m.)
28. AMENDING ENGINEERED MINICHROMOSOMES IN PLANTS
In planta Amendments
Zinc Finger NucleasesZinc Finger Nucleases
Durai et al., Nucleic Acids Res., 2005
29. Site-specific Recombination Systems
Cre/loxP system
Bacteriophage P1
Cre (Cyclization recombination) recombinase
loxP (locus of X-over of P1), 34-bp
Two directly repeated loxP sites - deletion
Two inverted loxP sites – inversion
Gilbertson, Trends Biotechnol., 2003
Fig-loxP Site
31. Zinc Finger Nucleases (ZFNs)
ZFNs are artificial RE generated by fusing a zinc finger DNA- binding domain to
a DNA-cleavage domain from FokI
Townsend et al., Nature,
2009
It can be engineered to target desired
DNA sequences
Non-specific cleavage domain: from
the type IIs RE FokI is used
ZF DNA-binding domain: recognize
between 9 and 18 bp
Uses: 1.Disabling an allele
2.Deletion of intervening sequences
32. (a) A diagrammatic engineered minichromosome generated by telomere-mediated chromosome truncation. The
minichromosome contains a terminal transgene locus (red ), which contains a promoter (Pro) driving expression of a
selection gene (S gene). The transgene cassette also contains a pair of directly oriented loxP sites flanking the selection
gene, as well as 3’ telomere repeat sequences (Telo). Crossing a line containing this minichromosome to one that
expresses Cre recombinase will lead to excision of the selection gene, and will leave a single wild-type loxP site. In a
subsequent transformation, a circular construct (donor molecule 1) containing a loxP site, an attP site (PP), a promoterless
S gene, a novel gene of interest (GOI-1), and two specific sites recognized by a zinc finger nuclease (ZF1) can be
introduced into the minichromosome-containing cell along with a plasmid expressing Cre recombinase.
Mechanisms To Amend Minichromosome Platforms
33. CONTD…
(b) The first novel gene has been added to the minichromosome transgene locus as a result of the previous Cre
recombination event. A specific zinc finger (targeting the ZF1 sites) could be used to delete the intervening sequences. In
a second round of gene stacking, a second donor plasmid (donor molecule 2) could be introduced into cells containing
the minichromosome along with an integrase enzyme that specifically leads to recombination between attP and attB sites.
In this round of integration, the donor molecule contains a promoterless selection gene, a second gene of interest (GOI-
2), novel attachment sites attB and attb (BB and bb, respectively), and a zinc finger nuclease recognition sequence.
Gaeta et al., Annu. Rev. Plant Biol., 2012
34. (c) The result of the previous integration event involves introduction of the second gene of interest. The result of
recombination between attachment sites BB and PP results in the site PB, which is not acted upon by the
integrase.
Similarly to panel b, a zinc finger nuclease could again be used to delete extraneous sequences. A third donor cassette
could be used and recombination could be targeted to the bb site (not shown). In each recombination event involving the
integrase, the target sites are destroyed, and thus attB and attP could be alternately used for sequential addition to the
minichromosome.
CONTD…
At any point, we can delete all sequences between the two loxP sites that flank
the entire cassette by crossing this line with another line that expresses Cre
recombinase
35. The artificial chromosome construction system can be used as powerful
mutagen
(Brown et al.,
2000)
It can also be used for gene stacking in plants, which is currently considered
as challenging for plant biotechnology
(Halpin., 2005)
Functional genomic studies could use minichromosomes as a platform for
adding specific genes or doses of chromosomal segments
Can be used in sequencing projects
POTENTIAL APPLICATIONS
36. AC platforms can provide solution to the stable expression & maintenance of
multiple transgenes in one genome
It represents a potential powerful research tool for understanding chromosome
structure & function
They can be useful for mass production of foreign proteins, pharmaceuticals, or
useful metabolites in plants
(Daniell et al., 2009)
The inheritance of multiple foreign genes as a unit
CONTD…
37. Next generation vectors for human gene therapy & plant genetic engineering
(Yu et al., 2007)
Can be used in site-specific recombination or retrofitting the minichromosomes
with additional foreign genes
(Ow., 2007)
AC could be easily introduced or removed from a genotype by genetic crosses
and would facilitate introgression of transgenes to different genetic backgrounds
It is possible that an important application of AC in plants will be combine them
with haploid breeding
(Ravi et al., 2010)
CONTD…
38. Summary Points…
Synthetic chromosomes in plants are likely to have more applications than in
other taxa because of the ease with which they can be manipulated throughout
their life cycle
Telomere-mediated chromosomal truncation works robustly in plants
Plant centromeres have an epigenetic component to their specification, which
constrains the approaches to producing engineered minichromosomes
Site-specific recombination systems and zinc finger nucleases provide means
to amend and grow synthetic plant chromosomes
39. It might be possible to develop a mini B chromosome-based genomic cloning
system for capturing large chromosome fragments
Site-specific recombination systems & zinc finger nucleases are valuable tools
for marker gene removal & gene targeting. Such technologies could be applied
to AC
The mechanism involved in de novo telomere formation (DNTF) & capping is
entirely unknown & are worthy for future study
Future Direction…Future Direction…
Editor's Notes
Retrotransposon: component of centromeres of a wide range of angiosperm species
implying that they play an important role in plant centromere evolution. constitutive transcription of this retrotransposon family.
Centromeres
The centromere is the region of each eukaryotic chromosome that attaches to spindle fibers during mitosis and meiosis. Proper centromere function is therefore very important for cell division and reproduction. Centromeres have been best studied in yeast, and all yeast centromeres have been shown to be remarkably similar to each other. Each centromere consists of three regions. The two flanking regions (regions I and III) are short, very highly conserved sequences (this means that virtually all of the centromeres have the same sequence, indicating that the particular sequence is important). Of these two regions, region III seems to be the most critical, and may be essential for binding to the spindle fibers. The central region is not highly conserved, but it is very rich in adenine and thymine - over 90% of the bases in this region are A or T!
Satellite DNA consists of very large arrays of tandemly repeating, non-coding DNA. Satellite DNA is the main component of functional centromeres, and form the main structural constituent of heterochromatin.[1][2]
The name &quot;satellite DNA&quot; refers to how repetitions of a short DNA sequence tend to produce a different frequency of the nucleotides adenine, cytosine, guanine and thymine, and thus have a different density from bulk DNA - such that they form a second or &apos;satellite&apos; band when genomic DNA is separated on a density gradient.
A repeated pattern can be between 1 base pair long (a mononucleotide repeat) to several thousand base pairs long, and the total size of a satellite DNA block can be several megabases without interruption. Most satellite DNA is localized to the telomeric or the centromeric region of the chromosome. The nucleotide sequence of the repeats is fairly well conserved across a species. However, variation in the length of the repeat is common. For example, minisatellite DNA is a short region (1-5kb) of 20-50 repeats. The difference in how many of the repeats is present in the region (length of the region) is the basis for DNA fingerprinting.
Satellite DNA, at least the microsatellite variety, is thought to have originated by slippage of a replicated chromosome against its template.
Centromeric retrotransposon in rice (CRR):
different CRR subfamilies may play different roles in the RNAi-mediated pathway for formation and maintenance of centromeric heterochromatin.
they play an important role in plant centromere evolution. In addition, their transcriptional activity is consistent with the notion that the transcription of centromeric retrotransposons has a role in normal centromere function.
In the budding yeast Saccharomyces cerevisiae, the functional centromere is defined by a 125-bp sequence (Clarke 1998).
Centromeric DNA sequences are not highly conserved, and their functional importance in many higher eukaryotes is still a matter of debate. the barley centromeric repeats are neither sufficient nor obligatory to assemble kinetochores.
The kinetochore, where specific proteins accumulate and mediate attachment to spindle fibers during nuclear divisions, is positioned at the centromere, hence at the primary constriction.
Neocentromere: A functional centromere in a novel location; may lack specific classes of deoxyribonucleic acid usually present in a centromere.Neocentromeres, new sites of functional kinetochore assembly, can form at ectopic loci because no DNA sequence is strictly required for assembly of a functional kinetochore.The knob heterochromatin sites in the chromosomes
will act as neocentromeres during meiosis in
the presence of abnormal chromosome 10, a variant
of the smallest chromosome in the maize complement
(RHOADES and VILKOMERSON, 1942)
Telomeres
Telomeres are the ends of the chromosomes. They consist of many tandem (head to tail) repeats of short (generally hexamer) sequences. In humans the telomeric sequence is TTAGGG. This sequence is repeated over 50 times at the end of each chromosome. These sequences are added to the ends of the chromosomes by an enzyme called telomerase. The reason telomeres exist is to provide stability to the chromosomal ends. As we&apos;ll see in the module on DNA replication, addition of telomeric sequences prevent the shortening of chromosomes that would otherwise occur during replication.
LEU2- REQUIRE LEUCINE.
A yeast artificial chromosome (YAC) is a vector used to clone DNA fragments larger than 100 kb and up to 3000 kb. YACs are useful for the physical mapping of complex genomes and for the cloning of large genes. First described in 1983 by Murray and Szostak, a YAC is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences named autonomous replicating sequence needed for replication and preservation in yeast cells. A YAC is built using an initial circular plasmid, which is typically broken into two linear molecules using restriction enzymes; DNA ligase is then used to ligate a sequence or gene of interest between the two linear molecules, forming a single large linear piece of DNA.[citation needed].
A typical YAC consists of centromere element (CEN) for chromosome segregation during cell division, telomere and origin of replication (ori) were isolated and joined on plasmid constructed in E.coli. For cloning purpose YAC is digested with restriction enzymes and recombinants are produced by inserting a large fragment of genomic DNA. This molecule can be maintained in yeast as YAC. [1] The transformant that contain YAC can be identified by red/white color selection. Non-transformed yeast contain white colonies. Red colonies of yeast contain the YAC molecule. Insertion of the DNA molecule into SnaB1 site, inactivates SUP4 its protein will not be expressed. This means colonies containing a YAC molecule with a DNA insert are in fact white.
Red colonies can therefore be excluded as they have no insert, growth on a medium without tryptophan or uracil will exclude colonies with no YAC vector. This is because the YAC vector encodes proteins that facilitate growth in the absence of these essential nutrients, whilst normal yeast are not able to grow in their absence.[2]
Advantages and Disadvantages
Yeast expression vectors, such as YACs, YIps (yeast integrating plasmids), and YEps (yeast episomal plasmids), have an advantage over bacterial artificial chromosomes (BACs) in that they can be used to express eukaryotic proteins that require posttranslational modification.
However, YACs are significantly less stable than BACs, producing &quot;chimeric effects&quot;: artifacts where the sequence of the cloned DNA actually corresponds not to a single genomic region but to multiple regions. Chimerism may be due to either co-ligation of multiple genomic segments into a single YAC, or recombination of two or more YACs transformed in the same host Yeast cell. [3]The incidence of chimerism may be as high as 50%. [4]. Other artifacts are deletion of segments from a cloned region, and rearrangement of genomic segments (such as inversion). In all these cases, the sequence as determined from the YAC clone is different from the original, natural sequence, leading to inconsistent results and errors in interpretation if the clone&apos;s information is relied upon. Due to these issues, the Human Genome Project ultimately abandoned the use of YACs and switched to bacterial artificial chromosomes, where the incidence of these artifacts is very low.Naturally yeast chro ranges from 230 to 1700 kb
A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli.[1][2] F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome&apos;s usual insert size is 150-350kbp.[3] A similar cloning vector called a PAC has also been produced from the bacterial P1-plasmid.
BACs are often used to sequence the genome of organisms in genome projects, for example the Human Genome Project. A short piece of the organism&apos;s DNA is amplified as an insert in BACs, and then sequenced. Finally, the sequenced parts are rearranged in silico, resulting in the genomic sequence of the organism.
Common gene components
oriS, repE - Ffor plasmid replication and regulation of copy number.parA and parBfor partitioning F plasmid DNA to daughter cells during division and ensures stable maintenance of the BAC.A selectable markerfor antibiotic resistance; some BACs also have lacZ at the cloning site for blue/white selection.T7 & Sp6phage promoters for transcription of inserted genes.[edit]Contribution to models of disease
[edit]Inherited disease
BACs are now being utilized to a greater extent in modelling genetic diseases, often alongside transgenic mice. BACs have been useful in this field as complex genes may have several regulatory sequences upstream of the encoding sequence, including various promoter sequences that will govern a gene&apos;s expression level. BACs have been used to some degree of success with mice when studying neurological diseases such as Alzheimer&apos;s disease or as in the case of aneuploidy associated with Down syndrome. There have also been instances when they have been used to study specific oncogenes associated with cancers. They are transferred over to these genetic disease models by electroporation/transformation, transfection with a suitable virus or microinjection. BACs can also be utilised to detect genes or large sequences of interest and then used to map them onto the human chromosome using BAC arrays. BACs are preferred for these kind of genetic studies because they accommodate much larger sequences without the risk of rearrangement, and are therefore more stable than other types of cloning vectors.[citation needed]
[edit]Infectious disease
The genomes of several large DNA viruses and RNA viruses have been cloned as BACs. These constructs are referred to as &quot;infectious clones&quot;, as transfection of the BAC construct into host cells is sufficient to initiate viral infection. The infectious property of these BACs has made the study of many viruses such as the herpesviruses, poxviruses and coronaviruses more accessible.[4][5][6]Molecular studies of these viruses can now be achieved using genetic approaches to mutate the BAC while it resides in bacteria. Such genetic approaches rely on either linear or circular targeting vectors to carry out homologous recombination.[7]
A human artificial chromosome (HAC) is a microchromosome that can act as a new chromosome in a population of human cells. That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small, roughly 6-10 megabases in size, and able to carry new genes introduced by human researchers. Yeast artificial chromosomes and bacterial artificial chromosomeswere created before human artificial chromosomes, which first appeared in 1997. They are useful in expression studies as gene transfer vectors and are a tool for elucidating human chromosome function. Grown in HT1080 cells, they are mitotically and cytogenetically stable for up to six months.
In mammalian systems, chromosomes have been engineered by various methods, including the top-down method (building chromosomes by truncation and modifications of endogenous chromosomes), and the bottom-up method (building chromosomes by assembling their component parts).
In plants, minichromosomes have been engineered by the top-down method. Studies with maize demonstrated that minichromosomes could be generated by adding plant telomere sequences to transgene cassettes (99, 100). Transformation with these constructs produced transgenic events on the termini
of truncated chromosomes via telomere mediated truncation.
Minichromosomes in higher eukaryotes can be generated via two approaches: by de novo assembly of an artificial minichromosome using appropriate sequences delivered into cells (“bottom-up” approach) or by the induced truncation or fragmentation of native chromosomes (“topdown” approach). The “bottom-up” approach has been used for assembly of a human artificial chromosome (HAC) by transfection of the cell line HT1080 with a mixture of
human centromere-specific alpha satellite, telomeric, and genomic carrier DNA (Harrington et al. 1997). Later, several groups reported the successful assembly of stable HACs using either cloned centromere-specific tandem repeats of alphoid DNA or large centromeric DNA segments (Grimes and Cooke 1998; Ikeno et al. 1998, 2002; Henning et al. 1999; Ebersole et al. 2000; Rudd et al. 2003; Basu et al. 2005a, b). However, for reasons which are not
yet clearly understood, reproducible assembly of HACs has only been accomplished in this one immortalized fibrosarcoma HT1080 cell line (Basu and Willard 2005). Analysis of HACs indicated that, upon delivery, DNA is probably subjected to rearrangements (multimerization, recombination, and/or amplification) resulting in artificial chromosomes which are much larger than the original constructs used for transformation (for review, see Lim and Farr 2004). In some clonal lines, HACs were unstable, resulting in loss of the minichromosome or integration into the host DNA (Shen et al. 2001; Rudd et al. 2003). In other cases, HACs were mitotically stable in the absence of selection for at least 9 months (Mejia et al. 2002). Recently, it has been demonstrated that HACs generated in HT1080 cells and subsequently transferred to mouse embryonic stem cells were mitotically stable, and further, could be successfully
transmitted to progeny in mice (Suzui et al. 2006).
We can produce A and B minichromosomes by telomere mediated truncation but they were more interested in B chromosome based minichromosomes, because B chromosomes has many interesting properties (Kato et al., 2005), such as: (i) the truncation of B chromosomes will not cause developmental or transmission problems as A chromosomes do; (ii) the B chromosome derivatives can be distinguished by their shape and the presence of a B chromosome specific repeat in and around the centromeric region; and (iii) the size of mini B chromosomes is not crucial because there will be no residual endogenous genes that might interfere with plant development and transgene expression. Recently, Carlson et al., (2007) developed maize minichromosomes (MMCs) and demonstrated that autonomous MMCs can be mitotically and meiotically maintained.
we were interested in B chromosome-based minichromosomes, because the B chromosome has many properties that make it preferable for engineered chromosomes. B chromosomes are naturally occurring supernumerary chromosomes (10–12). The B chromosome is basically inert, and its presence at low number does not affect the phenotype of plants. In addition, recombination of B chromosomes with the A set has never been observed; thus B chromosome-based vectors will have minimal detrimental effects on the host genome. The B chromosome of maize is basically inert, without any
known active genes (10–12). However, it is not known whether the lack of gene activity on B chromosomes is caused by the absence of genes or by suppression of transcription because of its heterochromatic nature.
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. Abbreviation: T-DNA, transfer DNA. (c) 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). Figure not drawn to scale.
Cre-lox: Bacteriophage P1
Flp-FRT: S. serevisiae
R-RS: Zygosaccharomyces rouxii
In 2009, Germany awarded the Max Delbrück medal to Dr. Klaus Rajewsky for his role in developing Cre-Lox recombination.
Cre-lox recombination could be used for conditional gene targeting in vivo.
Cre-Lox recombination is a site-specific recombinase technology widely used to carry out deletions, insertions, translocations and inversions in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems.
Cre recombinase
The Cre protein (encoded by the locus originally named as &quot;Causes recombination&quot;, with &quot;Cyclization recombinase&quot; being found in some references) [7][8] consists of 4 subunits and two domains: The larger carboxyl (C-terminal) domain, and smaller amino (N-terminal) domain. The total protein has 343 amino acids. The C domain is similar in structure to the domain in the Integrase family of enzymes isolated from lambda phage. This is also the catalytic site of the enzyme.
[edit]Lox P site
Lox P (locus of X-over P1) is a site on the Bacteriophage P1 consisting of 34 bp. There exists an asymmetric 8 bp sequence in between with two sets of palindromic, 13 bp sequences flanking it. The detailed structure is given below.
13bp8bp13bpATAACTTCGTATA -GCATACAT-TATACGAAGTTAT[edit]
2. FLP/frt system
Saccharomyces cerevisiae
FLP recombinase
frt (FLP recognition target), 34-bp
Two directed repeated frt sites - deletion
Two inverted frt sites - inversion
Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. DNA-cleavage domain
The non-specific cleavage domain from the type IIs restriction endonuclease FokI is typically used as the cleavage domain in ZFNs.[1] This cleavage domain must dimerize in order to cleave DNA[2]and thus a pair of ZFNs are required to target non-palindromic DNA sites. Standard ZFNs fuse the cleavage domain to the C-terminus of each zinc finger domain. In order to allow the two cleavage domains to dimerize and cleave DNA, the two individual ZFNs must bind opposite strands of DNA with their C-termini a certain distance apart. The most commonly used linker sequences between the zinc finger domain and the cleavage domain requires the 5&apos; edge of each binding site to be separated by 5 to 7 bp.[3]
Several different protein engineering techniques have been employed to improve both the activity and specificity of the nuclease domain used in ZFNs. Directed evolution has been employed to generate a FokI variant with enhanced cleavage activity that the authors dubbed &quot;Sharkey&quot;.[4] Structure-based design has also been employed to improve the cleavage specificity of FokI by modifying the dimerization interface so that only the intended heterodimeric species are active.[5][6][7][8]
[edit]DNA-binding domain
The DNA-binding domains of individual ZFNs typically contain between three and six individual zinc finger repeats and can each recognize between 9 and 18 basepairs. If the zinc finger domains are perfectly specific for their intended target site then even a pair of 3-finger ZFNs that recognize a total of 18 basepairs can theoretically target a single locus in a mammalian genome.
Various strategies have been developed to engineer Cys2His2 zinc fingers to bind desired sequences.[9] These include both &quot;modular assembly&quot; and selection strategies that employ either phage display or cellular selection systems.
The most straightforward method to generate new zinc-finger arrays is to combine smaller zinc-finger &quot;modules&quot; of known specificity. The most common modular assembly process involves combining three separate zinc fingers that can each recognize a 3 basepair DNA sequence to generate a 3-finger array that can recognize a 9 basepair target site. Other procedures can utilize either 1-finger or 2-finger modules to generate zinc-finger arrays with six or more individual zinc fingers. The main drawback with this procedure is the specificities of individual zinc fingers can overlap and can depend on the context of the surrounding zinc fingers and DNA. Without methods to account for this &quot;context dependence&quot;, the standard modular assembly procedure often fails unless it is used to recognize sequences of the form (GNN)N.[10]
Numerous selection methods have been used to generate zinc-finger arrays capable of targeting desired sequences. Initial selection efforts utilized phage display to select proteins that bound a given DNA target from a large pool of partially randomized zinc-finger arrays. More recent efforts have utilized yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells. A promising new method to select novel zinc-finger arrays utilizes a bacterial two-hybrid system and has been dubbed &quot;OPEN&quot; by its creators.[11] This system combines pre-selected pools of individual zinc fingers that were each selected to bind a given triplet and then utilizes a second round of selection to obtain 3-finger arrays capable of binding a desired 9-bp sequence. This system was developed by the Zinc-Finger Consortium as an alternative to commercial sources of engineered zinc-finger arrays.
(see: Zinc finger chimera for more info on zinc finger selection techniques)
[edit]Applications
Zinc finger nucleases have become useful reagents for manipulating the genomes of many plants and animals including arabidopsis,[12][13] tobacco,[14][15] soybean,[16] corn,[17] Drosophila melanogaster,[18] C. elegans,[19] sea urchin,[20] silkworm,[21] zebrafish,[22] frogs,[23] mice,[24] rats,[25] rabbits,[26] pigs,[27] cattle,[28] and various types of mammalian cells.[29] Zinc finger nucleases have also been used in a mouse model of haemophilia[30] and an ongoing clinical trial is evaluating Zinc finger nucleases that disrupt the CCR5 gene in CD4+ human T-cells as a potential treatment for HIV/AIDS. ZFNs are also used for the creation of a new generation of genetic disease models called isogenic human disease models.
[edit]Disabling an allele
ZFNs can be used to disable dominant mutations in heterozygous individuals by producing double strand breaks (DSBs) in the DNA (see Genetic recombination) in the mutant allele which will, in the absence of a homologous template, be repaired by non-homologous end-joining (NHEJ). NHEJ repairs DSBs by joining the two ends together and usually produces no mutations, provided that the cut is clean and uncomplicated. In some instances however, the repair will be imperfect, resulting in deletion or insertion of base-pairs, producing frame-shift and preventing the production of the harmful protein.[31] Multiple pairs of ZFNs can also be used to completely remove entire large segments of genomic sequence.[32] To monitor the editing activity, a PCR of the target area will amplify both alleles and if one contains an insertion, deletion, or mutation, it will result in a heterduplex single stranded bubble which cleavage assays such as Frontier Genomics&apos; SNiPerase-U or Transgenomics&apos; Surveyor Mutation Detection kits can easily detect. ZFNs have also been used to modify disease-causing alleles in triplet repeat disorders. Expanded CAG/CTG repeat tracts are the genetic basis for more than a dozen inherited neurological disorders including Huntington’s disease, myotonic dystrophy, and several spinocerebellar ataxias. It has been demonstrated in human cells that ZFNs can direct double-strand breaks (DSBs) to CAG repeats and shrink the repeat from long pathological lengths to short, less toxic lengths.[33]
Recently, a group of researchers have successfully applied the ZFN technology to genetically modify the gol pigment gene and the ntl gene in zebrafish embryo. Specific zinc-finger motifs were engineered to recognize distinct DNA sequences. The ZFN-encoding mRNA was injected into one-cell embryos and a high percentage of animals carried the desired mutations and phenotypes. Their research work demonstrated that ZFNs can specifically and efficiently create heritable mutant alleles at loci of interest in the germ line, and ZFN-induced alleles can be propagated in subsequent generations.
Similar research of using ZFNs to create specific mutations in zebrafish embryo has also been carried out by other research groups. The kdr gene in zebra fish encodes for the vascular endothelial growth factor-2 receptor. Mutagenic lesions at this target site was induced using ZFN technique by a group of researchers in US. They suggested that the ZFN technique allows straightforward generation of a targeted allelic series of mutants; it does not rely on the existence of species-specific embryonic stem cell lines and is applicable to other vertebrates, especially those whose embryos are easily available; finally, it is also feasible to achieve targeted knock-ins in zebrafish, therefore it is possible to create human disease models that are heretofore inaccessible.
[edit]Allele editing
ZFNs are also used to rewrite the sequence of an allele by invoking the homologous recombination (HR) machinery to repair the DSB using the supplied DNA fragment as a template. The HR machinery searches for homology between the damaged chromosome and the extra-chromosomal fragment and copies the sequence of the fragment between the two broken ends of the chromosome, regardless of whether the fragment contains the original sequence. If the subject is homozygous for the target allele, the efficiency of the technique is reduced since the undamaged copy of the allele may be used as a template for repair instead of the supplied fragment.
The first, and most important, problem is that the strategies of Harrington et al. and Ikeno et al. generated mini-chromosomes with no predictable relationship to the input DNA. Therefore, any gene that might be incorporated into this system would most likely be in an unpredictable sequence environment. But why does the DNA rearrange? There are two possible explanations: (1) it might need to assemble a structure that is large enough to be stable as a chromosome; or (2)The second problem is that although artificial chromosomes form following transfection, rearrangements frequently occur when the transfected DNA fails to form a new chromosome. Telomeric sequences often seed the formation of new telomeres when they are integrated into host chromosomes and this, in turn, leads to the loss of large segments of chromosomal DNA distal to the integration site8. The artificial chromosome construction systems are therefore powerful mutagens.
The third problem is that the absolute frequency of artificial chromosome formation is very low; minichromosomes formed at a frequency of ~531025 per
transfected cell when cationic lipids were used as a transfection reagent.
They can be increased readily in copy number and may aid in the mass production of foreign proteins, pharmaceuticals, or useful metabolites in plants (Daniell et al., 2009)
Adding transgenes to minichromosomes would avoid the disruption of endogenous genes by transgene integrations & would not be affected by position effects (Matzke et al., 1998)