SlideShare a Scribd company logo
Marker genes
Marker systems are tools for studying the transfer of genes into an experimental organism. In gene
transfer studies, a foreign gene, called a transgene, is introduced into an organism, in a process called
transformation. A common problem for researchers is to determine quickly and easily if the target
cells of the organism have actually taken up the transgene. A marker allows the researcher to
determine whether the transgene has been transferred, where it is located, and when it is expressed.
Marker genes exist in two broad categories:
I. Selectable marker genes and II. Reporter genes.
Selectable Marker Genes:
The selectable marker genes are usually an integral part of plant transformation system. They are
present in the vector along with the target gene. In a majority of cases, the selection is based on the
survival of the transformed cells when grown on a medium containing a toxic substance (antibiotic,
herbicide, antimetabolite). This is due to the fact that the selectable marker gene confers resistance to
toxicity in the transformed cells, while the non- transformed cells get killed.
A large number of selectable marker genes are available and they are grouped into three categories—
antibiotic resistance genes, antimetabolite marker genes and herbicide resistance genes.
(a) Antibiotic Resistance Genes
In many plant transformation systems, antibiotic resistance genes (particularly of E. coli) are used as
selectable markers. Despite the plants being eukaryotic in nature, antibiotics can effectively inhibit the
protein biosynthesis in the cellular organelles, particularly in chloroplasts.
Eg: Neomycin phosphotransferase II (npt II gene):
The most widely used selectable marker is npt II gene encoding the enzyme neomycin
phospho-transferase II (NPT II). This marker gene confers resistance to the antibiotic kanamycin. The
trans-formants and the plants derived from them can be checked by applying kanamycin solution and
the resistant progeny can be selected.
(b) Antimetabolite Marker Genes
Eg: Dihydrofolate reductase (dhfr gene)
The enzyme dihydrofolate reductase, produced by dhfr gene is inhibited by the antimetabolite
methotrexate. A mutant dhfr gene in mouse that codes for this enzyme which has a low affinity to
methotrexate has been identified. This dhfr gene fused with CaMV promoter results in a methotrexate
resistant marker which can be used for the selection of transformed plants.
(c) Herbicide Resistance Markers
Eg: Enolpyruvylshikimate phosphate synthase (epsps/aroA genes):
The herbicide glyphosate inhibits photosynthesis. It blocks the activity of enolpyruvylshikimate
phosphate (EPSP) synthase, a key enzyme involved in the biosynthesis of phenylalanine, tyrosine and
tryptophan. Mutant strains of Agrobacterium and Petunia hybrida that are resistant to glyphosate have
been identified. The genes epsps/aroA confer resistance to transgenic plants which can be selected.
Lists of selectable markers genes:
Reporter Genes:
A reporter gene may be regarded as the test gene whose expression can be quantified. The plant
transformation can be assessed by the expression of reporter genes. In general, an assay for the
reporter gene is carried out by estimating the quantity of the protein it produces or the final products
formed. A selected list of the reporter genes along with the detection assays is given in Table and
some of the important ones are discussed below.
β-Glucuronidase (GUS gene)
β-Glucuronidase producing gene (gus/uidA) is the most commonly used reporter gene in assessing
plant transformation for the following reasons:
i. β-Glucuronidase assays are very sensitive.
ii. Quantitative estimation of the enzyme can be done by fluorometric method (using substrate 4-
methylumbelliferryl P-D-glucuronide which is hydrolysed to 4-methylumbelliferone).
iii. Qualitative data on the enzyme can be obtained by histochemical means (enzyme localization can
be detected by chromogenic substance such as substrate X-gluc).
iv. No need to extract and identify DNA.
Green fluorescent protein (GFP gene)
Green fluorescent protein (GFP), coded by gfp gene, is being widely used in recent years. In fact, in
many instances, GFP has replaced GUS since assays of GFP are easier and non-destructive. Thus,
screening of even the primary transplants can be done by GFP which is not possible with other
reporter genes.
Gene for GFP has been isolated from jelly fish Aequorea victoria which is a luminescent organism.
The original gfp gene has been significantly modified to make it more useful as a reporter gene. GFP
emits fluorescence which can be detected under a fluorescent microscope.
Luciferase
1) Bacterial luciferase (luxA/luxB genes)
The bacterial luciferase genes (luxA and luxB) have originated from Vibrio harveyi. They can be
detected in some plant transformation vectors. The detection assay of the enzyme is based on the
principle of bioluminescence. Bacterial luciferase catalyses the oxidation of long-chain fatty
aldehydes that results in the emission of light which can be measured.
Firefly luciferase (luc gene)
The enzyme firefly luciferase, encoded by the gene luc, catalyses the oxidation of D-luciferin (ATP
dependent) which results in the emission of light that can be detected by sensitive luminometers. The
firefly luciferase gene, however, is not widely used as a marker gene since the assay of the enzyme is
rather cumbersome.

More Related Content

What's hot

Genomic library construction
Genomic library constructionGenomic library construction
Genomic library construction
Gurvinder Kaur
 
Site directed mutagenesis
Site  directed mutagenesisSite  directed mutagenesis
Site directed mutagenesis
Zain Khadim
 
Homologous Recombination (HR)
Homologous Recombination (HR)Homologous Recombination (HR)
Homologous Recombination (HR)
Raghav N.R
 
Agrobacterium mediated gene transfer
Agrobacterium mediated gene transferAgrobacterium mediated gene transfer
Agrobacterium mediated gene transfer
Rahulselvaraj
 
Methods of gene transfer in plant
Methods of gene transfer in plantMethods of gene transfer in plant
Methods of gene transfer in plant
Divya Srivastava
 
Restriction mapping
Restriction mappingRestriction mapping
Restriction mapping
Minali Singh
 
Library screening
Library screeningLibrary screening
Library screening
sridevi244
 
TRANSPOSON TAGGING
TRANSPOSON TAGGINGTRANSPOSON TAGGING
TRANSPOSON TAGGING
Mandeep Singh
 
Gene transfer methods @ujjwasirohi
Gene transfer methods @ujjwasirohiGene transfer methods @ujjwasirohi
Gene transfer methods @ujjwasirohi
ujjwal sirohi
 
Restriction-Modification system, Types of Restriction enzymes
Restriction-Modification system, Types of Restriction enzymesRestriction-Modification system, Types of Restriction enzymes
Restriction-Modification system, Types of Restriction enzymes
Kamaraj College of Engineering & Technology, Virudhunagar
 
Binary Vector, By KK Sahu sir
Binary Vector, By KK Sahu sirBinary Vector, By KK Sahu sir
Binary Vector, By KK Sahu sir
KAUSHAL SAHU
 
Agrobacterium mediated gene transformation
Agrobacterium mediated gene transformationAgrobacterium mediated gene transformation
Agrobacterium mediated gene transformation
awareswapnil1111
 
Callus and Suspension cultures
Callus and Suspension culturesCallus and Suspension cultures
Callus and Suspension cultures
srikaanth akshay
 
Electroporation
ElectroporationElectroporation
Electroporation
gowthamirajasekaran
 
Artificial chromosomes
Artificial chromosomesArtificial chromosomes
Artificial chromosomes
subhananthini jeyamurugan
 
Artificial chromosomes
Artificial chromosomesArtificial chromosomes
Artificial chromosomes
Darshana Ajith
 
Ri Plasmid
Ri PlasmidRi Plasmid
Ri Plasmid
egoistic_ek
 
Gene Transormation techniques
Gene Transormation techniquesGene Transormation techniques
Gene Transormation techniques
GauravRajSinhVaghela
 
Selectable marker genes
Selectable marker genesSelectable marker genes
Selectable marker genes
AakifahAmreen
 
Flavr savr tomato.ppt
Flavr savr tomato.pptFlavr savr tomato.ppt
Flavr savr tomato.ppt
Sakthivel R
 

What's hot (20)

Genomic library construction
Genomic library constructionGenomic library construction
Genomic library construction
 
Site directed mutagenesis
Site  directed mutagenesisSite  directed mutagenesis
Site directed mutagenesis
 
Homologous Recombination (HR)
Homologous Recombination (HR)Homologous Recombination (HR)
Homologous Recombination (HR)
 
Agrobacterium mediated gene transfer
Agrobacterium mediated gene transferAgrobacterium mediated gene transfer
Agrobacterium mediated gene transfer
 
Methods of gene transfer in plant
Methods of gene transfer in plantMethods of gene transfer in plant
Methods of gene transfer in plant
 
Restriction mapping
Restriction mappingRestriction mapping
Restriction mapping
 
Library screening
Library screeningLibrary screening
Library screening
 
TRANSPOSON TAGGING
TRANSPOSON TAGGINGTRANSPOSON TAGGING
TRANSPOSON TAGGING
 
Gene transfer methods @ujjwasirohi
Gene transfer methods @ujjwasirohiGene transfer methods @ujjwasirohi
Gene transfer methods @ujjwasirohi
 
Restriction-Modification system, Types of Restriction enzymes
Restriction-Modification system, Types of Restriction enzymesRestriction-Modification system, Types of Restriction enzymes
Restriction-Modification system, Types of Restriction enzymes
 
Binary Vector, By KK Sahu sir
Binary Vector, By KK Sahu sirBinary Vector, By KK Sahu sir
Binary Vector, By KK Sahu sir
 
Agrobacterium mediated gene transformation
Agrobacterium mediated gene transformationAgrobacterium mediated gene transformation
Agrobacterium mediated gene transformation
 
Callus and Suspension cultures
Callus and Suspension culturesCallus and Suspension cultures
Callus and Suspension cultures
 
Electroporation
ElectroporationElectroporation
Electroporation
 
Artificial chromosomes
Artificial chromosomesArtificial chromosomes
Artificial chromosomes
 
Artificial chromosomes
Artificial chromosomesArtificial chromosomes
Artificial chromosomes
 
Ri Plasmid
Ri PlasmidRi Plasmid
Ri Plasmid
 
Gene Transormation techniques
Gene Transormation techniquesGene Transormation techniques
Gene Transormation techniques
 
Selectable marker genes
Selectable marker genesSelectable marker genes
Selectable marker genes
 
Flavr savr tomato.ppt
Flavr savr tomato.pptFlavr savr tomato.ppt
Flavr savr tomato.ppt
 

Similar to Selectable markers and Reporter Genes.pdf

Screenable and Selectable Markers
Screenable and Selectable MarkersScreenable and Selectable Markers
Screenable and Selectable Markers
Shabnam Ameenudeen
 
Promoters
PromotersPromoters
Promoters
Msc2021
 
Gene transfer in plant-2.pptx
Gene transfer in plant-2.pptxGene transfer in plant-2.pptx
Gene transfer in plant-2.pptx
AbdullahAlJalil
 
reporter gene assays by Tahura Mariyam
reporter gene assays by Tahura Mariyam reporter gene assays by Tahura Mariyam
reporter gene assays by Tahura Mariyam
Tahura Mariyam Ansari
 
transgenics_17b.pptx
transgenics_17b.pptxtransgenics_17b.pptx
transgenics_17b.pptx
ridhi124788
 
Markers and reporter genes
Markers and reporter genesMarkers and reporter genes
Markers and reporter genes
RajDip Basnet
 
4. Applications of Biotechnology in Agriculture-II.pptx
4. Applications of Biotechnology in Agriculture-II.pptx4. Applications of Biotechnology in Agriculture-II.pptx
4. Applications of Biotechnology in Agriculture-II.pptx
EhtishamShah7
 
Use of transgenics in crop production
Use of transgenics in crop productionUse of transgenics in crop production
Use of transgenics in crop production
PragyaNaithani
 
Reporter genes
Reporter genesReporter genes
Reporter genes
SACHIN HALAPPA
 
Rahul ppt Gene Stacking.pptx
Rahul ppt Gene Stacking.pptxRahul ppt Gene Stacking.pptx
Rahul ppt Gene Stacking.pptx
Rahul Maurya
 
Engineering of secondary metabolism
Engineering of secondary metabolismEngineering of secondary metabolism
Engineering of secondary metabolism
Rahul Sen
 
Strain improvement-main
Strain improvement-mainStrain improvement-main
Strain improvement-main
hanifalpha
 
Prokaryotic Gene Regulation
Prokaryotic Gene RegulationProkaryotic Gene Regulation
Prokaryotic Gene Regulation
Jolie Yu
 
PHARMACOGENOMICS.pptx
PHARMACOGENOMICS.pptxPHARMACOGENOMICS.pptx
PHARMACOGENOMICS.pptx
ShivChandra16
 
TILLING & ECOTILLING
TILLING & ECOTILLINGTILLING & ECOTILLING
TILLING & ECOTILLING
Shivani Upadhyay
 
Reporter genes for the study of microbial ecology
Reporter genes for the study of microbial ecologyReporter genes for the study of microbial ecology
Reporter genes for the study of microbial ecology
Neha Sharma
 
transgenicplants-ppt-170509172214.pdf
transgenicplants-ppt-170509172214.pdftransgenicplants-ppt-170509172214.pdf
transgenicplants-ppt-170509172214.pdf
NamanSinha20
 
Transgenic plants ppt
Transgenic plants  pptTransgenic plants  ppt
Transgenic plants ppt
Kajol Roy
 
Lab Report Green Fluorescent Protein
Lab Report Green Fluorescent ProteinLab Report Green Fluorescent Protein
Lab Report Green Fluorescent Protein
Tina Jordan
 
marker free metheds.pptx
marker free metheds.pptxmarker free metheds.pptx
marker free metheds.pptx
ParthSharma157924
 

Similar to Selectable markers and Reporter Genes.pdf (20)

Screenable and Selectable Markers
Screenable and Selectable MarkersScreenable and Selectable Markers
Screenable and Selectable Markers
 
Promoters
PromotersPromoters
Promoters
 
Gene transfer in plant-2.pptx
Gene transfer in plant-2.pptxGene transfer in plant-2.pptx
Gene transfer in plant-2.pptx
 
reporter gene assays by Tahura Mariyam
reporter gene assays by Tahura Mariyam reporter gene assays by Tahura Mariyam
reporter gene assays by Tahura Mariyam
 
transgenics_17b.pptx
transgenics_17b.pptxtransgenics_17b.pptx
transgenics_17b.pptx
 
Markers and reporter genes
Markers and reporter genesMarkers and reporter genes
Markers and reporter genes
 
4. Applications of Biotechnology in Agriculture-II.pptx
4. Applications of Biotechnology in Agriculture-II.pptx4. Applications of Biotechnology in Agriculture-II.pptx
4. Applications of Biotechnology in Agriculture-II.pptx
 
Use of transgenics in crop production
Use of transgenics in crop productionUse of transgenics in crop production
Use of transgenics in crop production
 
Reporter genes
Reporter genesReporter genes
Reporter genes
 
Rahul ppt Gene Stacking.pptx
Rahul ppt Gene Stacking.pptxRahul ppt Gene Stacking.pptx
Rahul ppt Gene Stacking.pptx
 
Engineering of secondary metabolism
Engineering of secondary metabolismEngineering of secondary metabolism
Engineering of secondary metabolism
 
Strain improvement-main
Strain improvement-mainStrain improvement-main
Strain improvement-main
 
Prokaryotic Gene Regulation
Prokaryotic Gene RegulationProkaryotic Gene Regulation
Prokaryotic Gene Regulation
 
PHARMACOGENOMICS.pptx
PHARMACOGENOMICS.pptxPHARMACOGENOMICS.pptx
PHARMACOGENOMICS.pptx
 
TILLING & ECOTILLING
TILLING & ECOTILLINGTILLING & ECOTILLING
TILLING & ECOTILLING
 
Reporter genes for the study of microbial ecology
Reporter genes for the study of microbial ecologyReporter genes for the study of microbial ecology
Reporter genes for the study of microbial ecology
 
transgenicplants-ppt-170509172214.pdf
transgenicplants-ppt-170509172214.pdftransgenicplants-ppt-170509172214.pdf
transgenicplants-ppt-170509172214.pdf
 
Transgenic plants ppt
Transgenic plants  pptTransgenic plants  ppt
Transgenic plants ppt
 
Lab Report Green Fluorescent Protein
Lab Report Green Fluorescent ProteinLab Report Green Fluorescent Protein
Lab Report Green Fluorescent Protein
 
marker free metheds.pptx
marker free metheds.pptxmarker free metheds.pptx
marker free metheds.pptx
 

Recently uploaded

ISO/IEC 27001, ISO/IEC 42001, and GDPR: Best Practices for Implementation and...
ISO/IEC 27001, ISO/IEC 42001, and GDPR: Best Practices for Implementation and...ISO/IEC 27001, ISO/IEC 42001, and GDPR: Best Practices for Implementation and...
ISO/IEC 27001, ISO/IEC 42001, and GDPR: Best Practices for Implementation and...
PECB
 
How to Add Chatter in the odoo 17 ERP Module
How to Add Chatter in the odoo 17 ERP ModuleHow to Add Chatter in the odoo 17 ERP Module
How to Add Chatter in the odoo 17 ERP Module
Celine George
 
How to Setup Warehouse & Location in Odoo 17 Inventory
How to Setup Warehouse & Location in Odoo 17 InventoryHow to Setup Warehouse & Location in Odoo 17 Inventory
How to Setup Warehouse & Location in Odoo 17 Inventory
Celine George
 
BBR 2024 Summer Sessions Interview Training
BBR  2024 Summer Sessions Interview TrainingBBR  2024 Summer Sessions Interview Training
BBR 2024 Summer Sessions Interview Training
Katrina Pritchard
 
Leveraging Generative AI to Drive Nonprofit Innovation
Leveraging Generative AI to Drive Nonprofit InnovationLeveraging Generative AI to Drive Nonprofit Innovation
Leveraging Generative AI to Drive Nonprofit Innovation
TechSoup
 
Chapter 4 - Islamic Financial Institutions in Malaysia.pptx
Chapter 4 - Islamic Financial Institutions in Malaysia.pptxChapter 4 - Islamic Financial Institutions in Malaysia.pptx
Chapter 4 - Islamic Financial Institutions in Malaysia.pptx
Mohd Adib Abd Muin, Senior Lecturer at Universiti Utara Malaysia
 
writing about opinions about Australia the movie
writing about opinions about Australia the moviewriting about opinions about Australia the movie
writing about opinions about Australia the movie
Nicholas Montgomery
 
Présentationvvvvvvvvvvvvvvvvvvvvvvvvvvvv2.pptx
Présentationvvvvvvvvvvvvvvvvvvvvvvvvvvvv2.pptxPrésentationvvvvvvvvvvvvvvvvvvvvvvvvvvvv2.pptx
Présentationvvvvvvvvvvvvvvvvvvvvvvvvvvvv2.pptx
siemaillard
 
What is Digital Literacy? A guest blog from Andy McLaughlin, University of Ab...
What is Digital Literacy? A guest blog from Andy McLaughlin, University of Ab...What is Digital Literacy? A guest blog from Andy McLaughlin, University of Ab...
What is Digital Literacy? A guest blog from Andy McLaughlin, University of Ab...
GeorgeMilliken2
 
BÀI TẬP BỔ TRỢ TIẾNG ANH LỚP 9 CẢ NĂM - GLOBAL SUCCESS - NĂM HỌC 2024-2025 - ...
BÀI TẬP BỔ TRỢ TIẾNG ANH LỚP 9 CẢ NĂM - GLOBAL SUCCESS - NĂM HỌC 2024-2025 - ...BÀI TẬP BỔ TRỢ TIẾNG ANH LỚP 9 CẢ NĂM - GLOBAL SUCCESS - NĂM HỌC 2024-2025 - ...
BÀI TẬP BỔ TRỢ TIẾNG ANH LỚP 9 CẢ NĂM - GLOBAL SUCCESS - NĂM HỌC 2024-2025 - ...
Nguyen Thanh Tu Collection
 
C1 Rubenstein AP HuG xxxxxxxxxxxxxx.pptx
C1 Rubenstein AP HuG xxxxxxxxxxxxxx.pptxC1 Rubenstein AP HuG xxxxxxxxxxxxxx.pptx
C1 Rubenstein AP HuG xxxxxxxxxxxxxx.pptx
mulvey2
 
clinical examination of hip joint (1).pdf
clinical examination of hip joint (1).pdfclinical examination of hip joint (1).pdf
clinical examination of hip joint (1).pdf
Priyankaranawat4
 
Digital Artefact 1 - Tiny Home Environmental Design
Digital Artefact 1 - Tiny Home Environmental DesignDigital Artefact 1 - Tiny Home Environmental Design
Digital Artefact 1 - Tiny Home Environmental Design
amberjdewit93
 
Main Java[All of the Base Concepts}.docx
Main Java[All of the Base Concepts}.docxMain Java[All of the Base Concepts}.docx
Main Java[All of the Base Concepts}.docx
adhitya5119
 
LAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UP
LAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UPLAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UP
LAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UP
RAHUL
 
ANATOMY AND BIOMECHANICS OF HIP JOINT.pdf
ANATOMY AND BIOMECHANICS OF HIP JOINT.pdfANATOMY AND BIOMECHANICS OF HIP JOINT.pdf
ANATOMY AND BIOMECHANICS OF HIP JOINT.pdf
Priyankaranawat4
 
A Independência da América Espanhola LAPBOOK.pdf
A Independência da América Espanhola LAPBOOK.pdfA Independência da América Espanhola LAPBOOK.pdf
A Independência da América Espanhola LAPBOOK.pdf
Jean Carlos Nunes Paixão
 
Advanced Java[Extra Concepts, Not Difficult].docx
Advanced Java[Extra Concepts, Not Difficult].docxAdvanced Java[Extra Concepts, Not Difficult].docx
Advanced Java[Extra Concepts, Not Difficult].docx
adhitya5119
 
PCOS corelations and management through Ayurveda.
PCOS corelations and management through Ayurveda.PCOS corelations and management through Ayurveda.
PCOS corelations and management through Ayurveda.
Dr. Shivangi Singh Parihar
 
Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...
Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...
Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...
Dr. Vinod Kumar Kanvaria
 

Recently uploaded (20)

ISO/IEC 27001, ISO/IEC 42001, and GDPR: Best Practices for Implementation and...
ISO/IEC 27001, ISO/IEC 42001, and GDPR: Best Practices for Implementation and...ISO/IEC 27001, ISO/IEC 42001, and GDPR: Best Practices for Implementation and...
ISO/IEC 27001, ISO/IEC 42001, and GDPR: Best Practices for Implementation and...
 
How to Add Chatter in the odoo 17 ERP Module
How to Add Chatter in the odoo 17 ERP ModuleHow to Add Chatter in the odoo 17 ERP Module
How to Add Chatter in the odoo 17 ERP Module
 
How to Setup Warehouse & Location in Odoo 17 Inventory
How to Setup Warehouse & Location in Odoo 17 InventoryHow to Setup Warehouse & Location in Odoo 17 Inventory
How to Setup Warehouse & Location in Odoo 17 Inventory
 
BBR 2024 Summer Sessions Interview Training
BBR  2024 Summer Sessions Interview TrainingBBR  2024 Summer Sessions Interview Training
BBR 2024 Summer Sessions Interview Training
 
Leveraging Generative AI to Drive Nonprofit Innovation
Leveraging Generative AI to Drive Nonprofit InnovationLeveraging Generative AI to Drive Nonprofit Innovation
Leveraging Generative AI to Drive Nonprofit Innovation
 
Chapter 4 - Islamic Financial Institutions in Malaysia.pptx
Chapter 4 - Islamic Financial Institutions in Malaysia.pptxChapter 4 - Islamic Financial Institutions in Malaysia.pptx
Chapter 4 - Islamic Financial Institutions in Malaysia.pptx
 
writing about opinions about Australia the movie
writing about opinions about Australia the moviewriting about opinions about Australia the movie
writing about opinions about Australia the movie
 
Présentationvvvvvvvvvvvvvvvvvvvvvvvvvvvv2.pptx
Présentationvvvvvvvvvvvvvvvvvvvvvvvvvvvv2.pptxPrésentationvvvvvvvvvvvvvvvvvvvvvvvvvvvv2.pptx
Présentationvvvvvvvvvvvvvvvvvvvvvvvvvvvv2.pptx
 
What is Digital Literacy? A guest blog from Andy McLaughlin, University of Ab...
What is Digital Literacy? A guest blog from Andy McLaughlin, University of Ab...What is Digital Literacy? A guest blog from Andy McLaughlin, University of Ab...
What is Digital Literacy? A guest blog from Andy McLaughlin, University of Ab...
 
BÀI TẬP BỔ TRỢ TIẾNG ANH LỚP 9 CẢ NĂM - GLOBAL SUCCESS - NĂM HỌC 2024-2025 - ...
BÀI TẬP BỔ TRỢ TIẾNG ANH LỚP 9 CẢ NĂM - GLOBAL SUCCESS - NĂM HỌC 2024-2025 - ...BÀI TẬP BỔ TRỢ TIẾNG ANH LỚP 9 CẢ NĂM - GLOBAL SUCCESS - NĂM HỌC 2024-2025 - ...
BÀI TẬP BỔ TRỢ TIẾNG ANH LỚP 9 CẢ NĂM - GLOBAL SUCCESS - NĂM HỌC 2024-2025 - ...
 
C1 Rubenstein AP HuG xxxxxxxxxxxxxx.pptx
C1 Rubenstein AP HuG xxxxxxxxxxxxxx.pptxC1 Rubenstein AP HuG xxxxxxxxxxxxxx.pptx
C1 Rubenstein AP HuG xxxxxxxxxxxxxx.pptx
 
clinical examination of hip joint (1).pdf
clinical examination of hip joint (1).pdfclinical examination of hip joint (1).pdf
clinical examination of hip joint (1).pdf
 
Digital Artefact 1 - Tiny Home Environmental Design
Digital Artefact 1 - Tiny Home Environmental DesignDigital Artefact 1 - Tiny Home Environmental Design
Digital Artefact 1 - Tiny Home Environmental Design
 
Main Java[All of the Base Concepts}.docx
Main Java[All of the Base Concepts}.docxMain Java[All of the Base Concepts}.docx
Main Java[All of the Base Concepts}.docx
 
LAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UP
LAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UPLAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UP
LAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UP
 
ANATOMY AND BIOMECHANICS OF HIP JOINT.pdf
ANATOMY AND BIOMECHANICS OF HIP JOINT.pdfANATOMY AND BIOMECHANICS OF HIP JOINT.pdf
ANATOMY AND BIOMECHANICS OF HIP JOINT.pdf
 
A Independência da América Espanhola LAPBOOK.pdf
A Independência da América Espanhola LAPBOOK.pdfA Independência da América Espanhola LAPBOOK.pdf
A Independência da América Espanhola LAPBOOK.pdf
 
Advanced Java[Extra Concepts, Not Difficult].docx
Advanced Java[Extra Concepts, Not Difficult].docxAdvanced Java[Extra Concepts, Not Difficult].docx
Advanced Java[Extra Concepts, Not Difficult].docx
 
PCOS corelations and management through Ayurveda.
PCOS corelations and management through Ayurveda.PCOS corelations and management through Ayurveda.
PCOS corelations and management through Ayurveda.
 
Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...
Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...
Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...
 

Selectable markers and Reporter Genes.pdf

  • 1. Marker genes Marker systems are tools for studying the transfer of genes into an experimental organism. In gene transfer studies, a foreign gene, called a transgene, is introduced into an organism, in a process called transformation. A common problem for researchers is to determine quickly and easily if the target cells of the organism have actually taken up the transgene. A marker allows the researcher to determine whether the transgene has been transferred, where it is located, and when it is expressed. Marker genes exist in two broad categories: I. Selectable marker genes and II. Reporter genes. Selectable Marker Genes: The selectable marker genes are usually an integral part of plant transformation system. They are present in the vector along with the target gene. In a majority of cases, the selection is based on the survival of the transformed cells when grown on a medium containing a toxic substance (antibiotic, herbicide, antimetabolite). This is due to the fact that the selectable marker gene confers resistance to toxicity in the transformed cells, while the non- transformed cells get killed. A large number of selectable marker genes are available and they are grouped into three categories— antibiotic resistance genes, antimetabolite marker genes and herbicide resistance genes. (a) Antibiotic Resistance Genes In many plant transformation systems, antibiotic resistance genes (particularly of E. coli) are used as selectable markers. Despite the plants being eukaryotic in nature, antibiotics can effectively inhibit the protein biosynthesis in the cellular organelles, particularly in chloroplasts. Eg: Neomycin phosphotransferase II (npt II gene): The most widely used selectable marker is npt II gene encoding the enzyme neomycin phospho-transferase II (NPT II). This marker gene confers resistance to the antibiotic kanamycin. The trans-formants and the plants derived from them can be checked by applying kanamycin solution and the resistant progeny can be selected. (b) Antimetabolite Marker Genes Eg: Dihydrofolate reductase (dhfr gene) The enzyme dihydrofolate reductase, produced by dhfr gene is inhibited by the antimetabolite methotrexate. A mutant dhfr gene in mouse that codes for this enzyme which has a low affinity to
  • 2. methotrexate has been identified. This dhfr gene fused with CaMV promoter results in a methotrexate resistant marker which can be used for the selection of transformed plants. (c) Herbicide Resistance Markers Eg: Enolpyruvylshikimate phosphate synthase (epsps/aroA genes): The herbicide glyphosate inhibits photosynthesis. It blocks the activity of enolpyruvylshikimate phosphate (EPSP) synthase, a key enzyme involved in the biosynthesis of phenylalanine, tyrosine and tryptophan. Mutant strains of Agrobacterium and Petunia hybrida that are resistant to glyphosate have been identified. The genes epsps/aroA confer resistance to transgenic plants which can be selected. Lists of selectable markers genes:
  • 3. Reporter Genes: A reporter gene may be regarded as the test gene whose expression can be quantified. The plant transformation can be assessed by the expression of reporter genes. In general, an assay for the reporter gene is carried out by estimating the quantity of the protein it produces or the final products formed. A selected list of the reporter genes along with the detection assays is given in Table and some of the important ones are discussed below. β-Glucuronidase (GUS gene) β-Glucuronidase producing gene (gus/uidA) is the most commonly used reporter gene in assessing plant transformation for the following reasons: i. β-Glucuronidase assays are very sensitive. ii. Quantitative estimation of the enzyme can be done by fluorometric method (using substrate 4- methylumbelliferryl P-D-glucuronide which is hydrolysed to 4-methylumbelliferone). iii. Qualitative data on the enzyme can be obtained by histochemical means (enzyme localization can be detected by chromogenic substance such as substrate X-gluc). iv. No need to extract and identify DNA. Green fluorescent protein (GFP gene) Green fluorescent protein (GFP), coded by gfp gene, is being widely used in recent years. In fact, in many instances, GFP has replaced GUS since assays of GFP are easier and non-destructive. Thus, screening of even the primary transplants can be done by GFP which is not possible with other reporter genes.
  • 4. Gene for GFP has been isolated from jelly fish Aequorea victoria which is a luminescent organism. The original gfp gene has been significantly modified to make it more useful as a reporter gene. GFP emits fluorescence which can be detected under a fluorescent microscope. Luciferase 1) Bacterial luciferase (luxA/luxB genes) The bacterial luciferase genes (luxA and luxB) have originated from Vibrio harveyi. They can be detected in some plant transformation vectors. The detection assay of the enzyme is based on the principle of bioluminescence. Bacterial luciferase catalyses the oxidation of long-chain fatty aldehydes that results in the emission of light which can be measured. Firefly luciferase (luc gene) The enzyme firefly luciferase, encoded by the gene luc, catalyses the oxidation of D-luciferin (ATP dependent) which results in the emission of light that can be detected by sensitive luminometers. The firefly luciferase gene, however, is not widely used as a marker gene since the assay of the enzyme is rather cumbersome.