1. Reverse Genetics in Fisheries Research
Credit Seminar on
Reverse Genetics in Fisheries Research
Major Advisor
Dr. K Pani Prasad
Shyam K U
AAH-MA4-01

2. Introduction
Mendel started with his mutant phenotype to come with his rules.
Today, we know DNA sequences of many genes but not their phenotype.
Reverse Genetics is a term coined to desirable processes where information flows in opposite direction,
i.e., the gene is determined or altered directly and the resultant phenotype observed.
It discovers the normal role of cryptic DNA or protein sequences by mutating the sequence in vitro and looking for changes at the phenotypic level.

3. Forward Genetics Vs Reverse Genetics
Mutant Phenotype
Mutant Phenotype
Mutant Allele
Mutant Allele
FORWARD GENETICS
REVERSE GENETICS
DNA Sequence
DNA Sequence
Protein Sequence
Protein Sequence

4. Forward Genetics Vs Reverse Genetics
FG seeks to find the genetic basis of a phenotype or trait, RG seeks to find what phenotypes arise as a result of particular genetic sequences.
FG screens are intrinsically limited in their effectiveness to isolate mutations of every single gene due to functional redundancy between different genes and the need to have measurable phenotypes.
It is also quite time-consuming but without guarantees to identify the responsible mutated genes.

5. RG first identifies the gene of interest and subsequently obtain their mutations followed by the evaluation of the phenotypic effects of these mutations.
Can investigate the impact of induced variation in a specific gene on the organism.
RG is one of the modified application of recombinant DNA technology.
Important tools for RG are in vitro mutagenesis and the gene disruption (gene knock out)

6. Reverse Genetics Techniques
Large Scale Systematic Mutagenesis – Retrovirus mediated insertional mutagenesis,Tol2 Transposase system, Gene knock-out via TILLING
Gene Targeting by Site-Specific Genome Modification – Gene targeting via ZFNs, TALENs
Gene Knock-down via Morpholino nucleotides
4. Random Mutagenesis

7. Retrovirus Mediated Insertional Mutagenesis
Easiest way of expressing the desired gene by engineering the retrovirus via cloning.
Infecting embryonic stem cells with pseudo-typed retrovirus – integrates randomly into genome as a part of retroviral life cycle, causes the mutation.
A pseudo-typed MLV-retrovirus with its envelope protein replaced by G-protein of VSV shown infectivity and germline transmission in zebra fish embryos. (Lin et al., 1994; Wu et al., 2003; Wang et al., 2007).
A unique advantage of retroviral mutagenesis over chemical mutagens is that it allows rapid identification of mutated genes.

8. By using the retroviral sequence flanking the insertion site can be easily identified through inverse PCR or LM-PCR.
36748 essential genes for zebra fish have been successfully identified until (ZFIN Database., 2016; Amsterdam et al., 1999, 2004; Golling et al., 2002).
Limitations :- genes that share functional redundancy or do not create visible phenotypes during early development are usually ignored.
Its not practical for saturation mutagenesis screen due to its intensive labour and large fish maintenance requirement.

9. Injection of zebra fish embryo with retrovirus preparation
Raising of founder embryo generation
F1 generation by inbreeding/outbreeding of founder
Identification of F1 fish with multiple insertions
Raising F2 families by inbreeding selected F1 fishes
Sperms were collected and archived from 2nd and 3rd generation
DNA extracted from fin clips
Insertion sites were identified by LM-PCR and sequencing
Source : Huang et al., 2012

10. Tol2 transposon system
It’s a most common transgenic approach in zebra fish.
Tol2 transposable elements were originally discovered in the Japanese medaka, Oryzias latipes
Co-injection of expression constructs containing transgenes flanked by the Tol2 elements with the transposase mRNAs.
So, this will enhances the efficiency of trans-genesis and germ line transmission.

11. + Transposon construct in plasmid Transposase mRNA
Minimal Tol2 vector
Transposase mRNA +
Transposon construct in plasmid
mRNA translate to Transposase
Excision of construct from plasmid
Integration of construct into genomic DNA
Production of F1 generation & Detection by Green Fluorescence
Source: Kawakami K., 2007

12. TILLING
Targeting Induced Local Lesions IN Genome (TILLING) – 1st RG approach in Zebrafish
Combined method of forward and RG based on chemical mutagenesis to isolate mutants harboring point mutations in genes of interest.
TILLING - developed by the Henikoff laboratory to screen libraries of EMS- treated Arabidopsis for desired mutant alleles (Cobert et al., 2001; McCallum et al., 2000).
TILLING in zebrafish was applied by Wienholds in 2002 (Wienholds et al., 2002; Weinholds et al., 2003b)

13. The fishes are mutagenized by ENU (N-ethyl N-Nitrosourea), a chemical mutagen. (Zebrafish male)
Mutations in target genes (1 in 105 bp) are sought by sequencing the target regions.
So it requires large scale sequencing, difficulty to conduct as a routine technique.
Therefore community based mutation projects by TILLING were established by several consortia (eg: Sanger Institute – ).
Eg: TILLING for rag1 gene in zebrafish.

14. Zinc Finger Nucleases (ZFN)
ZFN are artificial endonuclease enzymes originally introduced as a hybrid restriction enzyme.
It consists of two domains - Zinc Finger domain – recognizes a specific sequence of genomic DNA & FokI restriction enzyme domain – cuts dsDNA.
ZFD will specifically bind a region where the FRED acts and FokI enzymes cuts the dsDNA region.
A dsDNA break is repaired by homologous recombination or a non- homologous end joining (NHEJ).

15. Protein Zif268 (Blue) containing three ZF in complex with DNA (Orange)
Zinc iron and coordinating amino acid (Green)

16. NHEJ is an error-prone process - It creates small deletion/insertion (indel) mutations in a site of lesion.
By engineering the zinc finger domains to bind specific loci of genomes, possible to introduce mutations into genes of interest.
Advantage : based on simple theory, viable tool for targeted mutagenesis.
Disadvantage : technically challenging and costly, potential risk of off-target effects, complexity regarding the designing of DNA.
Injected into fertilized eggs at the one or two cell stages
Individual harbouring mutations in a target gene are subsequently identified
These founder fish are outcrossed to obtain an F1 generation
Confirmed F1 incrossed and resulting F2 generation analyzed

17. TALEN Transcription Activator-Like Effector Nucleases (TALENs).
Same as ZFNs, two domains – restriction enzyme domain and DNA binding domain.
But in TALENs, the DNA binding domain is derived from plant pathogenic bacteria with TALE modules recognizing specific bases.
Advantages : more flexible and much simpler than ZFN.
Disadvantage : Off-target effects.

18. Source : Ed Davis, Technical Note, Genecopoeia

19. Site-Specific Genome Modification via ZFN and TALEN
A pair of mRNA encoding ZFN and TALEN are injected into embryo
restriction enzyme digestion, CEL I or T7 endonuclease digestion, sequencing analysis, melting curve analysis
Raising Mosaic embryos to founders
F1 generation (outcross between Founder and wild)
Fin clipping genotyping
Incross between F1 carriers
F2 generation and Phenotypic analysis
Source: Huang et al., 2012

20. Morpholino knockdown
Knockdown of gene function by morpholino antisense nucleotides -Most widely used RG technique in fish (zebrafish).
Morpholinos are chemically synthesized nucleotides with morpholine rings. This will resist the breakdown by nucleases.

21. Mostly they are designed to bind in the vicinity of the start codon to block initiation of translation or to splice acceptor sequences to cause aberrantly spliced mRNAs.
They are injected into freshly fertilized eggs and effectively block mRNA translation or splicing of gene up to 5dpf.
It can also be used to block the maturation of miRNA as well as to inhibit their binding to the target mRNA.
Advantages – easy to use and quick read out, high efficient.
Disadvantage – require dose optimization, off –target effects.

22. RG in context of Virology
Rescue of virus entirely from transfecting cloned cDNA plasmids encoding the viral components – Positional Cloning.
Ability to manipulate a cDNA intermediate, exact copy of the viral RNA genome, opens the possibility of deleting genes for studying their functions.
The use of recombinant DNA technology to convert viral genomes into complementary DNA and generate viruses from the cloned DNA.
Introducing targeted mutations to determine potential genetic factors of virulence or inserting heterologous genes of interest and using these recombinant viruses as gene vectors.

23. RG in History of Virology
Year
Event
Nature of Virus
Reference
1981
Recovery of poliovirus from cDNA
+ ve sense ss RNA
Racaniello and Baltimore, 1981
1994
Rescue of rhabdovirus rabies virus
- ve sense ss RNA
Schnell et al., 1994
1996
Rescue of trisegmented bunyavirus Bunyamwera virus
ve sense ss RNA
3 segments
Bridgen and Elliott., 1996
1998
Rescue of infectious pancreatic necrosis virus
segmented ds RNA
RNA virus infecting aquatic species
Yao and Vakharia., 1998
2000
Rescued synthetic salmonid rhabdovirus minigenomes
- ve sense ss RNA non-segmented
Biacchesi S et al., 2000
2001
Developed RG system for SJNNV
+ ve segmented ss RNA
Betanodavirus infecting fish
Iwamoto T et al., 2001
2006
Recovered a rec SAV from rainbow trout
Moriette C et al., 2006

24. Reverse Genetics System for Fish RNA viruses
First success in RG for a positive RNA virus is the Poliovirus (Picornaviridae family) was in 1981.
But after 13 years gap only RG system is used and revolutionized.
First description of segmented dsRNA virus recovery entirely from cDNA by Mundt and Vakharia for IBDV.
First RG system for a RNA virus infecting aquatic species is IPNV by Yao and Vakharia .

25. Fish RNA viruses RG system.
Novirhabdovirus
Infectious Haematopoietic Necrosis Virus (IHNV)
Viral Haemorrhagic Septicemia Virus (VHSV)
Alaphavirus
Sleeping Disease Virus (SDV)
Aquabirnavirus
Infectious Pancreatic Necrosis Virus (IPNV)
Nodavirus
Striped Jack Nervous Necrosis Virus (SJNNV)
Genome manipulations and rescue of + ve sense RNA viruses is easier.
But – ve sense RNA viruses require RNP complex for infectivity.

26. Source : Biacchesi S., 2011

27. Potential Applications
A powerful tool to study all the aspects of the virus biology and virus-host interaction.
Gives the opportunity to use the viruses as a live vaccines or as gene vectors.
Structure-Function study of individual viral genes….not by isolation in different systems (reductionist approach).
To study molecular basis of pathogenicity of virus.
To develop early, rapid and sensitive molecular diagnostics for emerging and existing viral pathogens.
To develop a animal model system for studying gene function and human disease research.

28. Boundaries for RG Experiments
Lethal mutations or mutations that are so detrimental that they are genetically unstable and cannot be easily examined.
A major constrain is how viruses are packaged ?
A more contentious issue is which viruses should be rescued
Potential thread of misuse and weapon for bioterrorism.
So, ethics based beneficial Reverse Genetics Research will always catalyze the modern research and its services are endless.

29. reference
Adams D M and Sekelsky J J., From sequence to phenotype: reverse genetics in Drosophila melanogaster., Nature Reviews Genetics 3:
Ahringer, J., ed. Reverse genetics.,WormBook, ed. The C. elegans Research Community, WormBook, doi/ /wormbook , 
Biacchesi S., 2011., The reverse genetics applied to fish RNA viruses., Veterinary Research 42: 12.
Bridgen A., Reverse Genetics of RNA viruses: Applications and perspectives., John Wiley and Sons ed. Introduction to Reverse Genetics. Pp 1-20.
Gilchrist E and Haughn G., Reverse genetics techniques: engineering loss and gain of gene function in plants., Briefings in Functional Genomics 9,2 :
Huang P., Zhu Z., Lin S., Zhang B., Reverse genetic approaches in Zebrafish., J. of Genetics and Genomics 39:
Lawson D N and Wolfe A S., Forward and reverse genetic approaches for the analysis of vertebrate development in the Zebrafish., Developmental Cell 21:
Lawson D N., Reverse genetics in Zebrafish: mutants, morphants and moving forward., Trends in Cell Biology 26, 2:
Pfaller K C., Cattaneo R., Schnell J M., Reverse genetics of Mononegavirales: how they works, new vaccines, and new cancer therapeutics., Virology :
Sugano Y. and Neuhauss C F S., Reverse genetics tools in Zebrafish: A forward dive into endocrinology., General and Comparative Endocrinology 188: