Gene tagging
To study the gene and its functions :
-Forward genetics: Involves random mutagenesis followed by screening to
recover mutants showing impairment for a particular biological processes.
First isolation of mutants………….then look for gene mapping and cloning.
-Reverse genetics: is the opposite approach where one starts with a cloned
gene whose function is unknown. This gene is mutated deliberately and the
effect analyzed.
Gene targeting
-
By homologous recombination, mutations can be introduced into predefined
genes ‘in vivo‘ known as gene targeting.
Gene tagging
-Gene tagging is the use of an insertional mutagen to mark interrupted genes
with a unique DNA sequence.
-This DNA sequence subsequently can be used as a target for hybridization or
as an annealing site for PCR primers, allowing flanking sequences to be
isolated.
Transposon tagging
-It is an insertion based technique.
-A piece of DNA is randomly inserted into the genome causing gene disruption
and loss of function.
-This causing gene may constitute a transposable element, i.e. a sequence that
can jump from site to site in the genome when supplied with the necessary
enzyme transposase or it may be a foreign DNA sequence that is introduced
into the cell.
Signature tagged mutagenesis (STM)
-The advantage of transposon tagging over traditional forms of mutagenesis is
that the interrupted gene becomes”tagged” with the insertion elements,
hence the strategy is sometimes termed Signature-tagged mutagenesis
(STM) .
Steps:
1.
Transposon tagging.
2.
Hybridization or PCR based techniques can be used to obtained the
flanking DNA..
3.
The sequence of the flanking DNA can then be used to interrogate
sequence databases, allowing the tagged gene to be associated with its
“parent” genomic clone phenotype.
4.
If insertion, also generates a mutant phenotype, the gene in the database
can then be ascribed a tentative function.
Note- Another advantage of insertional mutagenesis is insertion of element can
be modified into entrapment vector to provide information about the gene
it interrupts.
Gene tagging-Methodology
-It is the use of an insertional mutagen to mark interrupted genes with a unique
DNA sequence.
-Use this unique sequence as a target for hybridization or as an annealing site
for PCR primers…………allowing flanking sequences to be isolated.
-Use inverse PCR, in which primers annealing at each edge of insertion
element are designed to point outwards.
-By this way, if a genomic fragment is circularized, the PCR can be used to
amplify flanking regions…………..cloning and information about the
interrupted genes.
Modifications in tagging vectors
- Plasmid rescue vector
-In this the insertion vector contains the origin of replication and antibiotic resistance
gene from bacterial plasmid.
Methodology:
-Genomic DNA from tagged organism is digested with specific restriction enzyme that
does not cut in the insert.
-These resulting linear fragments are now self ligated and form circle.
-This complex mixture of circles now used to transform the bacteria under antibiotic
selection.
-Only the circle propagated will be having origin of replication and antibiotic resistance
genes, rest all the other circles lost.
-By this way, the genomic sequences flanking the insert can be isolated and selectively
amplified in a single step.
Note- It is more time consuming than the direct amplification by PCR.
-These “rescued plasmids” can be maintained as a permanent resource library.
Gene trap vector
-In this vector, the insertion element contains a visible marker gene such as lacZ
(encoding β-galactosidase) or a Gus A (Encoding β- glucuronidase) downstream of a
splice acceptor site.
Note- The marker gene will activate only when the element insert within the transcription
unit of a gene and generates a transcriptional fusion.
Limitations
-
However, gene trap strategy selects for insertions into genes and is very useful in
animals and plants with large amount of non-genic DNA.
-
Dependency on ‘in frame’ insertions, two-third of all “hits” on genes were not
recognized.
- Another thing is expression of marker relied only on the transcriptional activity of the
surrounding gene, and therefore, the non-expressed genes were not detected.
-
Soln
-The use of ribosome entry site by passed the need for in-frame design, it has greatly
increased the hit rate of gene traps.
-Use of second marker which is driven by its own promoter, but carries a downstream
splicing donor site……….this design making it dependent on the surrounding gene
for polyadenylation……………and therefore, it can be used for the detection of nonexpressed genes.
Enhancer trap technique
-
This construct comprises a visible marker gene downstream of a minimal promoter.
-
Interestingly, this minimal promoter is too weak to activate the marker gene and it is
not expressed. It expresses only when the construct integrates in the vicinity of an
endogenous enhancer, marker is activated and reports the expression profile driven
by enhancer.
-
It can be assess by some visible color products like blue if Lac Z used or ablation of
a group of cells if it drives a expression of toxin.
-
This technique mainly used for Drosophila model.
Activation tagging
-In this technique instead loss of function in response to insertion element, gain of
function takes place.
-The insertion element carries a strong outward-facing promoter and if, it integrates
adjacent to an endogenous gene, the gene will be activated by the promoter.
-This type of tagging causes over expression or ectopic expression of endogenous
gene.
Gene tagging by using Agrobacterium T-DNA or
plant transposons
-Plant transformations by A. tumefaciens involves the transfer of a small segment of
DNA , known as T-DNA from the plasmid harbored by tumefaciens.
-It means T-DNA acts as an insertional mutagen and can have been used for genome
wide mutagenesis programs.
-As T-DNA is not a transposon, it has no ability to ‘jump’ following
integration………..therefore, having advantage of generating stable insertions.
Features
-Gene insertion is essentially random.
-It affects only the gene, where it incorporated (within the gene space).
-Disadvantage is the tendency to generate complex, multicopy integration patterns and
sometimes deletions and gene rearrangements of surrounding genes.
-As Arabidopsis is a gene dense plant- having small introns, little intergenic space and
therefore, about 80 % genome is thought to represent genome.
By using various approaches of T-DNA tagging, presently about over 130,000 tagged
Arabidopsis lines are made available by University of Wisconsin known as
Arabidopsis knockout facility, which maintains a searchable database of mutant lines.
-
Many lines are generated by using simple T-DNA construct, activation tag, gene and
promoter traps in not only in arabidopsis, but also in rice.
-
Many are analyzed for reporter genes in seeds, flowers, while some are ubiquitous.
-
By using these lines containing T-DNA insertions have been generated and flanking
sequences have been characterized.
-
Strategy: Use gene specific primer and also the T-DNA specific primers ( if gene
sequence is not available , first sequence it).
Genome wide insertional mutagenesis in yeast: Use of
endogenous and heterologous transposons
-One strategy is use of Ty element as an insertional mutagen………..libraries of mutants
generated carrying Ty……used as “genetic foot print”.
As it is endogenous……….therefore, modified element with a unique DNA sequence
was used as a mutagen for unambigous identification of interrupted genes.
-A PCR based strategy was developed in which primer anneal with modified element
and also gene specific primers used to identify insertions at particular loci.
Disadvantage:
Ty based functional genomics studies had one disadvantage that it has tendency for the
element to insert at the 5’ end of genes transcribed by RNA Pol III i.e. mainly
transcribe tRNA genes, while protein coding genes are transcribed by RNA Pol II and
represent the vast majority of transcriptome.
The use of heterologous transposons in Yeast
-By using E.coli Tn3 transposon modified to make a gene trap vector.
Strategy:
Bacterial cell……………transformed by Yeast genomic construct……mutagenesis
carried out in surrogate environment……….genomic DNA reintroduced in
yeast……genome recombines with endogenous genome……….replacing the
existing sequence.
Bank of E.coli strains housing genomic DNA clones.
Advantage:
Stable insertions are generated, allowing the production of mutant lines as a permanent
resource.
E. coli transposons are not subject to the insertional bias of yeast Ty elements and
therefore, provide a better random coverage of genome.
Multifunctional E. coli Tn3 cassette for random
mutagenesis in yeast
-The cassette comprises Tn3 component, Lac Z, selectable marker and an epitope.
- The Lac Z gene and markers are flanked by Lox P (locus of crossover in P1) sites.
-Integration generates a mutant allele which may or may not be reveal a mutant
phenotype.
-The presence of the Lac Z gene at the 5’ end of the construct allows transcriptional
fusions to be generated………..so the insert can be used as a reporter construct to
reveal the normal expression profile of the interrupted gene.
If, Cre recombinase is provided, the LacZ gene and markers are deleted, leaving the
endogenous gene joined to the epitope tag, allowing protein localization to be
studied.
Note- reporter gene is downstream of a splice acceptor bracketed by subterminal loxP
sites……..allowing excision of most of the inserts following expression of Cre
recombinase. Outside the loxP sites, the transposon also carries an in-frame epitope
tag.
Therefore, following Cre mediated excision of the transposon, the endogenous gene
becomes fused to the epitope tag…….allowing characterization and localization of
proteins.
Genome wide insertional mutagenesis in vertebrates: Use of
artificial transposon systems
-It was observed that approx 5 % of all naturally occurring recessive mutations in the
mouse are caused by a particular family of retroviruses when they integrate into or
adjescent to an endogenous gene.
-By using retroviral gene trap vector many ES lines has been generated and flanking
sequences had been determined to generate a database of gene trap sequence
tags.
Direct application of retrovirus based vectors in whole vertebrate animals is more
challenging due to lack of active retro-transposon in vertebrate genomes.
-Vertebrate genomes contain millions of Mariner-type transposons, but not active due to
the accumulation of mutation…..by using the assumption that different mutations
would occur in different copies of the element, and that a consensus sequence
should represent an active, archetypal element……..by looking at endogenous fish
Mariner-type retrotransposons in the same manner…………….’Sleeping
Beauty’………is used for large scale mutagenesis studies in fish as well as in
mammals.
Similarly, Frog prince is a reconstructed amphibian retro transposon based on
endogenous non-functional elements in the frog Rana pipens.