The document discusses several strategies for removing selectable marker genes from transgenic plants while retaining the gene of interest:
1. Site-specific recombination-based strategies use recombinases like Cre/lox to excise the selectable marker gene flanked by recombinase recognition sites.
2. Transposon-based strategies use transposable elements like Ac/Ds to mobilize and separate the selectable marker from the gene of interest.
3. Homologous recombination-based strategies induce double-strand breaks that can be repaired through homologous recombination, replacing the selectable marker with the desired gene.
marker free transgenic, strategies to developed marker free transgenic, commonly used marker, Positive selection system, Negative selection system,Abiotic stress related genes as selection markers, Avoiding the use of selectable marker genes, Strategies to eliminate marker genes from transgenic, Co-transformation,Site-specific recombination, Multi-auto transformation vector, Intra chromosomal recombination system,Transposition system, screenable markers
In plant and animal biotechnology, we used marker genes as selection of our GOI in host organism, but there are some problems related o these marker genes. Here we discussed about some marker free mathedologies.
marker free transgenic, strategies to developed marker free transgenic, commonly used marker, Positive selection system, Negative selection system,Abiotic stress related genes as selection markers, Avoiding the use of selectable marker genes, Strategies to eliminate marker genes from transgenic, Co-transformation,Site-specific recombination, Multi-auto transformation vector, Intra chromosomal recombination system,Transposition system, screenable markers
In plant and animal biotechnology, we used marker genes as selection of our GOI in host organism, but there are some problems related o these marker genes. Here we discussed about some marker free mathedologies.
Introduction
◦ The process of developing transgenic plants without the
presence of selectable markers (or) by use of more acceptable
marker genesis regarded as Clean Gene Technology.
◦ Also called “Marker Free Approach” for transgenic plant.
Need of CGT
◦ The products of some marker genes may be toxic or allergic.
◦ The antibiotic resistance might be transferred to pathogenic
microorganisms in the soil.
◦ There is a possibility of creation of superweeds that are resistant
to normally used herbicides.
Methods of CGT
◦1. Co-Transformation
◦2. Site Specific Recombination
◦3. Transposon‐Based Marker Method
◦4. Intra Chromosomal Homologous Recombination
1.Co-transformation
◦ Co-transformation is a method for production of marker free
transformants based on Agrobacterium- or biolistic mediated
transformation in which a SMG and gene of interest are on
separate constructs.
◦ SMGs can subsequently be removed from the plant genome
during segregation and recombination that occurs during sexual
reproduction by selecting on the transgene of interest and not
the SMG in progeny.
2. Site Specific Recombination
◦ Also called “mediated marker deletion”.
◦ The deletion of short, recognized DNA sequence with the activity of
recombinase enzymes has been a major step to acquire the marker free
transgenic plant.
◦ Systems for removal of marker gene
1. Cre/loxP: 2. FLP/FRT: 3. R/RS:
E.coli Bacteriophage Saccharomyces cerevisiae Zygosaccharomyces
3. Transposon‐based marker method
◦ This process is quite similar to site specific recombination where
transposons or jumping genes are used instead of a
recombination and recognition sites.
◦ Steps:
•(i) Insertion of the marker gene onto a transposon, a segment
of DNA that “hops” around in the plant’s genome;
•(ii) co-transformation with gene of interest
•(iii) Segregation of the marker gene.
4. Intrachromosomal recombination system
◦Non‐recombinase
◦ Spontaneous excision
attp: attachment P region of bacteriophage λ
ICRS
◦ Recombination sites are engineered into the plant, but no
recombinase is expressed.
◦Intrachromosomal recombination in plants is obtained by
insertion of SMG between two direct repeats of attP that
facilitates spontaneous excision.
◦ Base composition of the attP site sequence is A + T rich, which is
conjectured to play a recombination-stimulating role.
.Conclusion and future prospects
◦ The removal of marker gene from the transgenic plants supports multiple
transformation cycles for transgene pyramiding.
◦ It is clear that several viable methods for the removal of unwanted marker
genes already exist.
◦ It seems highly likely that continued work in this area will soon remove the
question of publicly unacceptable marker genes.
◦ At present there is no commercialization of marker free transgenic crop.
◦ But development of marker free transgenics would further increase the crop
improvement program.
Reporter stem cell lines are valuable models which enable noninvasive, live monitoring of marker onset and expression in a cell-specific manner. Some methods have been used to derive such cell lines based on lineage promoter-driven reporter expression.
Gene reporter is a valuable in vitro tool that facilitates live monitoring and tracking a cell type of interest. Lineage reporters are either created using minimal lineage-specific promoter-driven reporter systems or via knock-in of the reporter into the endogenous promoter region. Choice of the reporter is critical based on intended application.
New advances in genome-editing technologies have lowered these barriers for creating knock-in reporter lines.
https://www.creative-biogene.com/support/genome-editing-generation-of-reporter-stem-cells.html
Process whereby a marker is used for indirect selection of a genetic determinant or determinants of a trait of interest (i.e. productivity, disease resistance, abiotic stress tolerance, and/or quality).
Trait of interest is selected not based on the trait itself but on a marker linked to it.
The assumption is that linked allele associates with the gene and/or quantitative trait locus (QTL) of interest. MAS can be useful for traits that are difficult to measure, exhibit low heritability, and/or are expressed late in development.
Pre-Requisites: Two pre-requisites for marker assisted selection are: (i) a tight linkage between molecular marker and gene of interest, and (ii) high heritability of the gene of interest.
Markers Used: The most commonly used molecular markers include amplified fragment length polymorphisms (AFLP), restriction fragment length polymorphisms (RFLP), random amplified polymorphic DNA (RAPD), simple sequence repeats (SSR) or micro satellites, single nucleotide polymorphisms (SNP), etc. The use of molecular markers differs from species to species also.
Current trends in pseduogene detection and characterizationShreya Feliz
This presentation gives the insight of the current trends in detecting and characterizing Pseudogenes. Pseudogenes detection by bioinformatics may enhance the understanding of Pseudogenes and take research to the next step.
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.
Introduction
◦ The process of developing transgenic plants without the
presence of selectable markers (or) by use of more acceptable
marker genesis regarded as Clean Gene Technology.
◦ Also called “Marker Free Approach” for transgenic plant.
Need of CGT
◦ The products of some marker genes may be toxic or allergic.
◦ The antibiotic resistance might be transferred to pathogenic
microorganisms in the soil.
◦ There is a possibility of creation of superweeds that are resistant
to normally used herbicides.
Methods of CGT
◦1. Co-Transformation
◦2. Site Specific Recombination
◦3. Transposon‐Based Marker Method
◦4. Intra Chromosomal Homologous Recombination
1.Co-transformation
◦ Co-transformation is a method for production of marker free
transformants based on Agrobacterium- or biolistic mediated
transformation in which a SMG and gene of interest are on
separate constructs.
◦ SMGs can subsequently be removed from the plant genome
during segregation and recombination that occurs during sexual
reproduction by selecting on the transgene of interest and not
the SMG in progeny.
2. Site Specific Recombination
◦ Also called “mediated marker deletion”.
◦ The deletion of short, recognized DNA sequence with the activity of
recombinase enzymes has been a major step to acquire the marker free
transgenic plant.
◦ Systems for removal of marker gene
1. Cre/loxP: 2. FLP/FRT: 3. R/RS:
E.coli Bacteriophage Saccharomyces cerevisiae Zygosaccharomyces
3. Transposon‐based marker method
◦ This process is quite similar to site specific recombination where
transposons or jumping genes are used instead of a
recombination and recognition sites.
◦ Steps:
•(i) Insertion of the marker gene onto a transposon, a segment
of DNA that “hops” around in the plant’s genome;
•(ii) co-transformation with gene of interest
•(iii) Segregation of the marker gene.
4. Intrachromosomal recombination system
◦Non‐recombinase
◦ Spontaneous excision
attp: attachment P region of bacteriophage λ
ICRS
◦ Recombination sites are engineered into the plant, but no
recombinase is expressed.
◦Intrachromosomal recombination in plants is obtained by
insertion of SMG between two direct repeats of attP that
facilitates spontaneous excision.
◦ Base composition of the attP site sequence is A + T rich, which is
conjectured to play a recombination-stimulating role.
.Conclusion and future prospects
◦ The removal of marker gene from the transgenic plants supports multiple
transformation cycles for transgene pyramiding.
◦ It is clear that several viable methods for the removal of unwanted marker
genes already exist.
◦ It seems highly likely that continued work in this area will soon remove the
question of publicly unacceptable marker genes.
◦ At present there is no commercialization of marker free transgenic crop.
◦ But development of marker free transgenics would further increase the crop
improvement program.
Reporter stem cell lines are valuable models which enable noninvasive, live monitoring of marker onset and expression in a cell-specific manner. Some methods have been used to derive such cell lines based on lineage promoter-driven reporter expression.
Gene reporter is a valuable in vitro tool that facilitates live monitoring and tracking a cell type of interest. Lineage reporters are either created using minimal lineage-specific promoter-driven reporter systems or via knock-in of the reporter into the endogenous promoter region. Choice of the reporter is critical based on intended application.
New advances in genome-editing technologies have lowered these barriers for creating knock-in reporter lines.
https://www.creative-biogene.com/support/genome-editing-generation-of-reporter-stem-cells.html
Process whereby a marker is used for indirect selection of a genetic determinant or determinants of a trait of interest (i.e. productivity, disease resistance, abiotic stress tolerance, and/or quality).
Trait of interest is selected not based on the trait itself but on a marker linked to it.
The assumption is that linked allele associates with the gene and/or quantitative trait locus (QTL) of interest. MAS can be useful for traits that are difficult to measure, exhibit low heritability, and/or are expressed late in development.
Pre-Requisites: Two pre-requisites for marker assisted selection are: (i) a tight linkage between molecular marker and gene of interest, and (ii) high heritability of the gene of interest.
Markers Used: The most commonly used molecular markers include amplified fragment length polymorphisms (AFLP), restriction fragment length polymorphisms (RFLP), random amplified polymorphic DNA (RAPD), simple sequence repeats (SSR) or micro satellites, single nucleotide polymorphisms (SNP), etc. The use of molecular markers differs from species to species also.
Current trends in pseduogene detection and characterizationShreya Feliz
This presentation gives the insight of the current trends in detecting and characterizing Pseudogenes. Pseudogenes detection by bioinformatics may enhance the understanding of Pseudogenes and take research to the next step.
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.
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Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
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1. Chapter-6:Elimination selective marker
from trangenic plants
Following the production of transgenic plants, the
selectable marker gene(s) used in the process are
redundant,and their retention may be undesirable.
Several strategies can be used to remove selectable
markers from transgenic plants, while retaining the gene of
interest (GOI).
1
2. The strategies that can be used to
remove selectable markers from transgenic
plants the following :
site-specific recombination based
Transposon based systems
Intrachromosomal recombination
system based
Transformation without selection
Co-transformation of a marker gene
Homologous recombination
2
3. site-specific recombination
site-specific recombination based method
in which the transformation cassette
comprises, in addition to the GOI, the
selectable marker flanked by specific
recombinase recognition sites.
Microbial site-specific recombinases have
the ability to cleave DNA at specific sites
and ligate it to the cleaved DNA at a
second target sequence.
3
4. The excision of foreign DNA that is placed in
between recognition sites in a direct repeat
orientation has been used to eliminate
unwanted transgenic material from the nuclear
genome of plants.
The most used recombination systems are:
Cre/lox from bacteriophage P1
FLP/FRT from Saccharomyces cerevisiae and
R/RS from Zygosaccharomyces rouxii[.
4
5. These systems are belonging to the tyrosine recombinase
family .
After the reaction, a recombination site (lox, FRTor RS) is
remaining in the genome and it could potentially serve as a
site for integrative recombination.
However, re-insertion of the elimination fragment has not
been detected , probably because excision is an
intramolecular event, whereas integration needs
interaction between unlinked site and second, the excised
circle cannot replicate autonomously and is probably
rapidly lost in vivo
5
6. The site-specific recombination systems can be
divided in two categories according to the position of
the recombinase gene:
The recombinase gene and the selectable
marker are on a different vector and the
recombinase gene is delivered to the plant
containing the SMG by re-transformation or
by sexual crosses .
The selectable marker and the recombinase
genes are on the same vector between the
recombination sites
6
7. .
Figure :Removal of selectable marker genes through site specific recombinases.
7
8. In a second category of methods using site-specific
recombination, the selectable marker and the recombinase
genes are on the same vector between the recombination sites
This system is often referred to as “auto-excision” or self-excision
.
The auto-excision strategy is a versatile system that could be
applied in every species and that shows flexibility in spatial
and temporal control.
The expression of the recombinase gene can be induced by either
external or intrinsic signals resulting in auto-excision of both the
recombinase and marker genes placed within the excision site
boundaries after their function is no longer needed.
The control of excision is enabled by the regulated promoter used to
control the recombinase gene.
8
10. Limitation of both system
they require a time-consuming and labor-intensive breeding step,
and
they are only applicable to sexually reproducing species or some
species where the retransformation is available.An alternative
approach depends on the expression of the recombinase transiently
with a modified plant virus carrying the cregene (PVX-Cre); as well
as in kanamycin resistant tobacco with aTMV-Cre .This method can
be applied to vegetatively propagated and long life cycle plants, but
the lack of virus-based transformation system in these species is a
drawback that should be improved in the future.
that Cre can catalyze recombination between certain naturally
occurring “pseudo-loxsites” that can be highly divergent from
the loxconsensus sequence.
This toxic effect was also investigated in creexpressing transgenic
plants where a correlation was found between aberrant phenotypes
and constitutive creexpression
10
14. Transposon based systems
This an approach in which the selectable marker gene
is flanked by sequences recognized by a transposase,
so that when the cassette is introduced, the selectable
marker is mobilized to a new location in the genome,
thereby becoming separated from the GOI.
Controlling elements may be grouped into families.The
members of each family may be divided into two classes:
autonomous elements or non-autonomous elements.
14
16. Autonomous elements have the ability to transpose whereas
the non-autonomous elements are stable (but can transpose
with the aid of an autonomous element through trans-
activation)
Two mutations:
dissociation (Ds) : The Ds element is
located on chromosome 9and it has the
effect of causing chromosome breakage
at a point on the chromosome adjacent
to its location
activator (Ac):Ac is capable of autonomous
move ment, but Ds moves only in the
presence of Ac.
16
18. Transposable elements (e.g. Ac/Ds from maize) can mediate
repositioning of genetic material in the plant genome.
The Ac/Dstransposable element system has been used for relocation
and elimination of a selectable marker in tomato and rice].
Transposable elements can be excised from the genome after the
expression of the transposase; they can either re-insert or not (Fig.
2).Taking into account these options, two approaches can be
followed. In the first one, if one counts on re-insertion of the
transposon, the gene of interest (GOI) is placed on the transposable
element.Thus, the GOI will be excised and can be reinserted in a
locus that is not linked to the locus in which the selectable marker
gene is located; they can be segregated in the next generation
[47, 49]. In a second approach, one relies on the fact that the
transposon will not be re-inserted [50].An example of such a
system is the one described by Ebinuma et al. [51], in which
the iptselectable marker gene was inserted in an Acderivative.
However, marker-free transgenic pl
18
23. Draw backs:
The transposition efficiency is variable in different species.
The method is labor intensive and time consuming
because it requires crossing transgenic plants and the
selection of the progeny
The method shows low efficiency of marker gene
elimination because of the tendency of transposable
elements to reinsert in positions genetically linked to the
original position.
The systems are the genomic instability of transgenic plants
because of the continuous presence of heterologous transposons
and the generation of mutations because of insertion and excision
cycles. Transposition can induce genome rearrangements, including
deletions, inverted duplications, inversions, and translocations
The system cannot be used for sterile plants and
vegetative propagated species and is impractical for plants
with a long life cycle.
23
26. Transformation without selection
The most straightforward method to obtain marker-free plants is to
transform without any selectable marker gene.
However, most of the transformation protocols described are inefficient
and just few cells integrate the foreign DNA. Nonetheless, some groups
have studied the feasibility to obtain transgenic plants omitting
selection.
De Buck et al. failed to obtain any transgenic plants
when Arabidopsisroots were transformed via A. tumefaciensand shoots
regenerated on non-selective media. However, in tobacco protoplast
transformation, these authors obtained a total transformation
frequency of 18%.Transformation protocols have important influence
on these and other results. For example, in a study
where Arabidopsiswas transformed by the floral dip protocol and
seedlings were grown on non-selective media, transgenic plants could
be obtained with an efficiency of 3.5%
26
27. Co-transformation of a marker gene
The SMG and the gene of interest can be delivered
by:
(i) twodifferent Agrobacteriumstrains each
containing a binary plasmid carrying a single
T-DNA region
(ii) a single Agrobacteriumstrain, either
containing one plasmid with two separate T-
DNAs (Fig. 1C) [or
(iii) containing two separate plasmids each
containing a T-DNA
27
28. co-transformation can be achieved by particle co-
bombardment .
The co-transformation strategy is limited
because :
co-integration of both T-DNAs at the same
genomic locus is frequently observed leading
to linkage between the marker and the
transgene, which makes their segregation
impossible.
This phenomenon has even more frequently
been observed with particle bombardment-
mediated transformation.
28
29. The methods cannot be applied to
sterile plants and vegetatively
propagated species, and are not
practical in plants with a long life
cycle such as trees
The approach requires the generation
of many transformants (to find unlinked
marker genes and genes-of-interest) and
Crossing steps (to remove the marker
gene) making it a labor intensive work.
29
32. Homologous recombination
This method is based the DNA repair machinery of plant
cells. Indeed, efficient repair of double-strand breaks (DSBs)
is important for survival of all organisms.
DSBs can be repaired via :
homologous recombination (HR) or
non-homologous end-joining (NHEJ) .
The ratio of HR to NHEJ events increases if homologous
sequences near the brake are available .
During the repair process the gene can be converted or
deleted.
32