This document discusses gene trapping as a method for identifying novel plant genes. It describes how gene trap vectors containing a reporter gene and selectable marker can disrupt genes when inserted into a plant genome. This leads to expression of the reporter gene under control of the trapped gene's regulatory elements. The document outlines different types of gene trap vectors, including enhancer, promoter, gene and polyA traps. It also discusses considerations for gene trap design such as choice of reporter gene, insertion vehicle, and generation of trapped lines. An example case study of an Arabidopsis gene trapping project is provided.

1. GENE TRAPS FOR PL ANT
DEVELOPMENT AND
GENOMICS
COURSE INSTRUCTOR :
DR. V.K. SHARMA
DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY & MOLECULAR BIOLOGY
Dr . RAJENDRA PRASAD CENTRAL AGRICULTURAL UNIVERSITY
PUSA ,SAMASTIPUR, (BIHAR)- 848125
PRESENTED BY:
KANCHAN YADAV
MSc. AGRIL.
BIOTECHNOLOGY

2. CLASSICAL GENETIC APPROACH
This approach to gene identification rely on
disruption of a gene leading to a
recognizable phenotype
BUT NOT ALL GENES CAN BE UNCOVERED
BY MUTAGENESIS !!
FIRSTLY, MANY GENES ARE FUNCTIONALLY
REDUNDANT, SHARING OVERLAPPING
FUNCTIONS WITH OTHER GENES
SECOND, MANY GENES FUNCTION AT
MULTIPLE STAGES OF DEVELOPMENT

3. GENE TRAPPING-BETTER APPROACH
• Gene trapping is a unique
method that helps to identify
novel gene by producing
random gene disruption by
inserting a DNA element,
contains a reporter gene and a
selectable marker, throughout
the genome.

4. STRATEGY FOR GENE TRAPPING
CONSTRUCT OF GENE TRAP
VECTORS
CHOICE OF INSERTION
VEHICLE
CHOICE OF REPORTER GENE

5. TRAP VECTORS
•Should provide a reporter as a tag to easily
detect endogenous gene expression
•And should allow rapid identification of the
trapped gene by serving as a molecular tag.
•No mutant phenotype is required

6. TYPES
Enhancer trap
vectors
Promoter trap
vectors
Secretory trap
vectorsPoly A trap vectors
Gene trap vectors

7. REGULATORY COMPONENTS OF A GENE -
IMPORTANT FOR ITS EXPRESSION
Enhancer a set of short sequence elements which stimulate transcription of a
gene.
Promoter a combination of short sequence elements to which RNA polymerase
binds in order to initiate transcription of a gene
Polyadenylation addition of typically 200 A residues to the 3' end of a mRNA.
The poly(A) tail is important for stabilizing mRNA.

8. ENHANCER TRAP VECTOR STRATEGY
NOT SUFFICIENT TO DRIVE
EXPRESSION OF REPORTER
GENE
INSERTED NEAR A CIS
ACTING ENHANCER ELEMENT
EXPRESSION OF REPORTER GENE
UNDER CONTROL OF
NEIGHBOURING CHROMOSOMAL
ENHANCER
VECTOR CONTAINING REPORTER GENE
+ SELECTABLE MARKER + POLY A SEQUENCE + WEAK PROMOTER

9. HOW IT WORKS
Investigate how and when enhancer DNA sequences can affect gene
regulation, and also aid in the determination of their possible location

10. ADVANTAGES AND DISADVANTAGES
Can effectively trap enhancer sequences.
May not be easy to identify and locate the enhancer causing the
specific expression pattern
It shows a 5’ bias,
Technique is not very mutagenic.
Requires screening of a very large populations
Able to trap only active genes ,not the transcriptionally silent
genes

11. PROMOTER TRAP VECTOR STRATEGY
VECTOR CONTAINING PROMOTERLESS REPORTER GENE + SELECTABLE
MARKER + POLY A SEQUENCE
INSERTED INTO EXONIC REGION OF ENDOGENOUS ACTIVE GENE IN
CORRECT ORIENTATION
TRANSCRIPTION OF REPORTER GENE UNDER CONTROL OF PROMOTER
AND ENHANCER OF INTERRUPTED ACTIVE GENE

12. HOW IT WORKS

13. ADVANTAGES AND DISADVANTAGES
 Used to study tissue-specific, developmental stage-specific and stress-induced
promoters
Method has high mutagenicity rate as compared to enhancer trap
Reporter expression requires both transcriptional fusion and in-frame,
translational fusion with a coding region of the interrupted gene.
Frequency with which promoter-trap vectors insert into exons is exceedingly
low
Insertion in intronic regions results in complete degradation of reporter
mRNA.

14. GENE TRAP VECTORS
VECTORS CONTAINING 5’ SPLICE ACCEPTOR (splicing site at the end of
intron)+ PROMOTERLESS REPORTER GENE + SELECTABLE MARKER +
POLY A SEQUENCE
INTEGRATION OF VECTOR IN EXONIC OR INTRONIC REGION
RESULTS IN PRODUCTION OF FUSION PROTEIN

15. HOW IT WORKS
A gene trap vector can successfully trap promoter, splicing
apparatus, exons and introns

16. ADVANTAGES AND DISADVANTAGES
This technique has high mutagenicity and trapping efficiency
Can detect insertion events in both exons and introns
Presence of IRES in certain vectors helps in translation of the reporter without in
frame fusion with the gene.
Reporter gene can disrupt the gene activity (i.e. produce inactive proteins)
Insertion could be lethal to the cell/organism
It can cause mutation only when inserted in correct orientation.
It cannot trap transcriptionally inactive genes

17. POLY A TRAP VECTORS

18. ADVANTAGES AND DISADVANTAGES
Can trap genes that are not expressed in undifferentiated
cells.
Can successfully trap 3’ region of the gene.
Shows a 3’ bias.
This bias tends to limit the mutagenicity of the insertions

19. SECRETORY TRAP VECTOR
 DETECT INTEGRATION IN GENES THAT
ENCODE CELL SURFACE PROTEINS
 CONTAIN TRANSMEMBRANE PORTION
FUSED TO REPORTER GENE
 GENES WHICH HELPS ITS
INCORPORATION IN THE MEMBRANE
FOLLOWING INSERTION IN GENES
THAT ENCODE CELL SURFACE
 IT CAN DISTINGUISH BETWEEN NO
INSERTION AND INSERTION IN NON
SECRETORY GENE
TRANSMEMBRANE
PORTION
MEMBRANE GENE –
ACTIVE REPORTER
GENE
NON SECRETORY
GENE – INACTIVE
REPORTER GENE

20. CHOICE OF REPORTER GENE
GUS – Bacterial gene uidA encodes a soluble β- glucuronidase
enzyme which breaks down glucuronide substrate to give a
coloured reaction
GFP – This gene has been obtained from jellyfish
Aequorea Victoria
It emits green fluorescent light in the blue to ultraviolet range
Luc- Luciferase isolated from the North American firefly
,Photinus pyralis catalyzes the oxidative decarboxylation of
luciferin to oxyluciferin . Luminescense in terms of light
production can be recorded in a luminometer

21. CHOICE OF INSERTION VEHICLE
T DNA
Allow quick generation of large fragments of
independent insertions, also not site specific and
genomic T DNA insertions are generally stable
TRANSPOSABLE
ELEMENTS
Low copy number ,remobilization is readily
possible

22. CREATION OF GENE TRAPPED LINES
Identification of insertion events
Genetic screen

23. • The transformed cells are selected on the basis of selectable marker.
• The commonly used selectable markers in gene trapping are
neomycin phosphotransferase II(NPTII or
neo),
kanamycin resistance gene(KAN),
hygromycin phophotransferase (hptI)
puromycin resistance (PURO) genes
IDENTIFICATION OF INSERTION
EVENTS

24. GENETIC SCREEN
After screening of the progeny for stable reporter gene assay, the trapped lines are established
as hypomorphic, loss of function and gain of function allele.
A genetic screen of mutants based solely on
the expression pattern of the mutated gene
is termed as expression screen.
A genetic screen of mutants based solely on
the sequence of the mutated gene is the
genotypic screen.
It is done by reporter gene assay,
expression-trapping screen, induction
trapping, FACS etc.
It is done by using various variants of
polymerase chain reaction. Most common is
reverse transcriptase PCR . It involves
5’RACE, 3’RACE, INVERSE PCR, ADAPTOR
PCR, TAIL PCR etc.

25. CASE STUDY

26. CASE STUDY CONTD.
• To monitor the expression of T-DNA-tagged
plant genes in vivo, a collection of 20,261
transgenic lines of Arabidopsis were
generated with the promoter trap vector
pTluc , which carries a promoterless firefly
luc (luciferase) reporter gene linked to the
right T-DNA border.
• By detection of bioluminescence in 3-week-
old seedlings, 753 lines were identified
showing constitutive, organ-specific, and
stress-responsive luciferase expression
patterns.
• To facilitate the identification of well-
defined luciferase expression patterns, a
pooled seed stock was established. Several
lines showed sugar, salt, and abscisic acid
(ABA)-inducible luciferase activity.

27. CASE STUDY CONTD.
• Segregation analysis of 215 promoter trap
lines indicated that about 50% of plants
contained single insertions, whereas 40%
carried two and 10% carried three or more T-
DNA tags.
• Sequencing the T-DNA insert junctions
isolated from 17 luciferase-expressing lines
identified T-DNA tags in 5- and 3-transcribed
domains and translational gene fusions
generated by T-DNA insertions in exons and
introns of Arabidopsis genes.

28. CASE STUDY CONTD.
• Tissue specific expression of eight wild-type Arabidopsis genes was confirmed to be
similar to the luminescence patterns observed in the corresponding luciferase –tagged
lines.
• Here , they described the characterization of a transcriptional luc reporter gene fusion
with the WBC-type ABC transporter gene At1g17840.
• Expression of wild-type and luciferase-tagged At1g17840 alleles revealed similar
induction by salt ,glucose, and ABA treatments and gibberellin – mediated down-
regulation of ABA-induced expression.

29. GENE TRAPS IN PLANTS

30. LIST OF PROMOTER ISOLATED AND
MUTATED BY GENE TRAP IN PLANTS
S.No. Promoter Specificity of expression Source species Reference
1. A1EM Embryo Arabidopsis Topping et al. 1994
2. Cryptic Seed coat specific Nicotiana tabacum Fobert et al.1994
3. HVT1 Tapetum and vascular tissue Arabidopsis Wel et al.1997
4. Pyk20 Nematode fonding structure Arabidopsis Puzio et al.1998
5. tcup Constitutive Nicotiana tabacum Foster et al.1999
6. Cryptic Guard cell Arabidopsis Pleach et al.2000
7. Cryptic Roots Arabidopsis Moher et al.2000
8. Lj Gbp 1 Roots Lotus japonicus Webb et al.2000
9. Eif.4A 1 Growing tissues, young leaves Arabidopsis De Grave et
10. EXORDIUM(EXO) Meristematic cells Arabidopsis Farrar et al.2003

31. ADVANTAGES
Powerful tool to characterize novel genes and analyze their
importance in biological phenomena.
A rapid and cost-efficient method that is ideally suited for large-scale
mutagenesis and full-genome exploration.
Trapping generates a single-cell reporter of transcriptional activity,
rather than assessing messenger RNA (mRNA) abundance in a cell
population.
Functional studies may easily be accomplished directly on the
trapped cells or on organisms derived from them.

32. ADVANTAGES
The expression pattern of the trapped gene can be followed by monitoring
reporter gene activity.
Reporter expression has also been used to study environmental and
hormonal responsive genes.
Genes expressed at very low levels can also be identified.
Due to their small size gene trap vectors change the genomic context of the
trapped gene only minimally and a faithful expression pattern is obtained.
Gene trap insertions create null alleles in most cases.

33. LIMITATIONS
• Does not always generate null alleles
• Trapping is not entirely random
• Phenotypes of the mutants are unpredictable
• It cannot be used for genes which are permanently
switched off
• Multiple copies of the trap vector can be integrated.
• The particular gene of interest may not be mutated by
this method.

34. CONCLUSION
The ability to efficiently trap sequence and detect the
expression of genes, regardless of their transcriptional
activity, has made gene trapping an exceptional tool for
gene discovery.

35. REFERENCES
• Springer, P.S. (2000) Gene Traps: Tools for Plant Development and Genomics, The Plant Cell, Vol. 12,
1007–1020.
• Kumari ,A., Sharma, V. K. And Kumar ,H.(2018) Gene Trapping: A Powerful tool of functional genomics
to identify novel genes, International Journal of Genetics, Vol. 10,325-332.
• Martha , C. A., Laura, M. Z., Izabella, K., Agnes, C., Csaba K., and Laszlo M.S.(2004). Gene Trapping with
Firefly Luciferase in Arabidopsis. Tagging of Stress-Responsive Genes ,Plant Physiology,Vol.134,18-27.
• Yamamoto, Y.Y., Tsuhara , Y., Gohda K., Suzuki K. and Matsui M. (2003) Plant Journal, Vol.35, 273–283.
• Lee, T., Shah, C. and Xu ,E.Y. (2007) Molecular Human Reproduction, Vol. 13, 771–779.
• Campisi ,L., Yang Y., Heilig, E., Herman, B., Cassista , A.J., Allen, D.W., Xiang, H. and Jack ,T. (1999) Plant
Journal ,Vol. 17, 699-707.
• He, Y., Tang, W., Swain ,J. D., Green, A. L., Jack, T. P. and Gan ,S. (2001) Plant Physiology , Vol. 126, 707-
716.