2013

Andrijana Gjeorgieva
UIST Saint Paul the Apostle
26-Dec-13
Reverse genetics engineering

Contents
Introduction ............................................................................................................................................ 3
Techniques of reverse genetics ................................................................................................................ 4
1. Site-directed mutagenesis................................................................................................................ 4
Site-directed mutagenesis using M13 phage .................................................................................... 4
2. Gene silencing ................................................................................................................................. 6
Reverse engineering in C.elegans ..................................................................................................... 7
3. TILLING ............................................................................................................................................ 8
TILLING Phytophthora ...................................................................................................................... 8
4. Gene disruption by homologues recombination ............................................................................... 9
Gene targeting ............................................................................................................................... 10
Protein engineering ....................................................................................................................... 10
Cancer therapy .............................................................................................................................. 11
Conclusion ............................................................................................................................................. 11










Introduction
Imagine a brave new world where we will have all the species that gone extinct, a world where there
will not be diseases anymore, no hunger and no deaths. A big picture of a family of species put down on
one ground. Pretty much of a chaos, right? Well, it can be true if things go out of the path that a human
being will set it for them before. Some things are not meant to be as they werent with the species that
gone extinct and the ones that came after, or those that prolonged their lives within the years and now.
With the technology and the knowledge we have gathered starting centuries ago, we can now attempt
to build or create all of those lost things or, try to reduce or totally get rid of the various diseases that
are spread around the globe.
Genetics is the study of the genes, their structure and function, science of hereditary and variation in
living beings. We have two approaches in this branch of sciences; on one side is the forward genetics
and on the other, reverse genetics. No matter how opposite they may seem, both of them aim to
determine the gene function.
Forward genetics or forward genetic screen deals with the determination of a gene or collection of
genes responsible for a particular phenotype. The reverse genetics, on the other hand, deals with
already known gene and examine the effects of its disruption on the phenotypes.









We now have whole genomes sequenced and many researchers have access to them. Having a
sequence of the genome is the first step or lead of the reverse genetics process. Starting from it, the
scientists can try to determine the function of a particular gene and its product. This gene of interest is
modified or disrupted and the effects showed by its phenotype are screened afterwards.

Techniques of reverse genetics

1. Site-directed mutagenesis
Site-directed mutagenesis (SDM), sometimes called site-specific mutagenesis, is a process that produces
mutations in DNA that are controlled by us. Simply said, we can decide what mutations we want and
then we will produce them. This means that we can change, not only the base sequence of DNA and
genetic code of a gene, but also the gene product, the protein. We can decide to change a particular
amino acid in a polypeptide chain of a protein/enzyme by changing the codon in the gene coding for
that amino acid. Changing the amino acid will often change the properties of the protein/enzyme, but
not always for the better as we expect. This is one of the techniques that can be used in protein
engineering - one of the most sophisticated applications of recombinant DNA technology - where the
properties of a protein, such as an enzyme, are altered in an attempt to 'improve' it by changing
(mutating) the gene coding for the protein using Site-Directed Mutagenesis. Desired improvements
might be increased thermostability, altered substrate range, reduction in negative feedback inhibition,
altered pH range, etc.
Site-directed mutagenesis using M13 phage
M13 is a small filamentous E. coli bacteriophage cloning vector which behaves like a plasmid for part of
its replication cycle. It is rather unusual in that it produces both double-stranded (dsDNA) and single-
stranded DNA (ssDNA) in different phases of its replication cycle. When in replicative form (RF) inside
the host cell, M13 acts rather like a plasmid, the DNA being double stranded (ds), but, when packaged
up in capsids to form phage particles to be released from the host cell, the DNA is single stranded. M13
(unlike lambda phage in its lytic cycle) does not cause lysis and death of the E. coli host cells. Instead,
infected host cells excrete M13 phage particles containing ssDNA in large numbers.
Stages of SDM by using M13:
1. Isolate required enzyme gene, e.g. via mRNA and its conversion into cDNA.
2. Sequence the DNA of the gene (in order to decide on change required for primer in stage 5).
3. Splice gene into M13 vector dsDNA and transduce E. coli host cells.
4. Isolate ssDNA in phage particles released from host cells.
5. Synthesize an oligonucleotide primer with the same sequence as part of the gene but with altered
codon (mismatch/mispair) at desired point(s). For example, one of the codons in DNA coding for the
amino acid Alanine is CGG. If the middle base is changed by SDM from G to C the codon sequence
becomes CCG which codes for a different amino acid (Glycine).
6. Mix oligonucleotide with recombinant vector ssDNA, carried out at low temperature (0-10oC) and in
high salt concentration to allow hybridization between oligonucleotide and part of gene. Under these
low stringency conditions hybridization occurs despite the mismatch. Remember that complementarity
between two pieces of ssDNA does not have to be 100% for hybridization to occur if stringency is low.
7. Use DNA polymerase to synthesize remainder of strand. (Oligonucleotide acts as a primer for the DNA
synthesis.) Then add ligase to make join between primer and new strand permanent dsDNA molecule.

1. Site-directed mutagenesis using M13 phage
8. Transform E. coli cells and allow them to replicate recombinant vector molecule.
9. DNA replication is semi-conservative, therefore two types of clone are produced each of which
excretes phage particles containing ssDNA:
Type 1: contain the wild-type gene (i.e. unaltered)
Type 2: contain the mutated gene.
Ratio of the two types should be 1:1 but is not usually because E. coli "edits out" some of the
mismatches.
10. Select mismatch clones bearing the mutation, using the same oligonucleotide (which originally
served as a primer in Stages 5 and 6) as a probe (add a label to it). The probe must now be used under
conditions of high stringency (rather than low stringency as it was when used as a primer) to ensure that
it only hybridizes with the altered DNA sequence of the desired clones and not with the DNA of clones
containing the wild-type gene.
11. If gene expression is required, use these clones to extract mutated DNA and insert it into an
expression vector system with appropriate promoter, etc. to produce the modified gene product
('designer protein').

2. Gene silencing
Gene silencing is a general term used to describe the epigenetic regulation of gene expression. This term
refers to the ability of a cell to prevent the expression of a certain gene. Gene silencing can occur during
either transcription or translation and is often used in research. In particular, methods used to silence
genes are being increasingly used to produce therapeutics to combat cancer and diseases, such as
infectious diseases and neurodegenerative disorders. Gene silencing is considered a gene knockdown
mechanism since the methods used to silence genes, such as RNAi, generally reduce the expression of a
gene by at least 70%, but do not completely eliminate it.
We have three types or units by which the gene silencing is classified:
Type 1: Transcriptional gene silencing (RNAi)>
- Genomic Imprinting
- Paramutation
- Transposon and transgene silencing
- Transcriptional silencing
- RNA-directed DNA methylation
Type 2: Post-transcriptional gene silencing>
- RNA Interference
- Nonsense mediated decay
Type 3: Meiotic gene silencing>
- Transvection
- Meiotic silencing of unpaired DNA

RNA interference (RNAi) is the process by which expression of a target gene is inhibited by antisense and
sense RNAs (The strand of the dsRNA that is identical in sequence to a region in target mRNA molecule is
called the sense strand, and the other strand which is complimentary is the antisense strand) by
destruction of specific mRNA molecules. It works based on the ability of double-stranded sequences to
recognize and degrade sequences that are complementary to them. RNAi has been used for a systematic
analysis of gene function in C. elegans by generating loss of function phenotypes, creating a library of
worms expressing dsDNA corresponding to different genes. Genome-wide RNAi screens against these
libraries of predicted genes have allowed study of a variety of biological processes in C. elegans.
One difficulty in using RNAi as a reverse genetic technique is that throughput is limited by the ability to
deliver siRNAs to target loci. It is also labor intensive, can give ambiguous results, and can be unsuitable
for isolating mutants that have lethal or sterile phenotypes.

Reverse engineering in C.elegans
Caenorhabditis elegans is a free-living (non-sessile), transparent nematode (roundworm) that lives in
temperate soil environments. C. elegans was the first multicellular organism to have its genome
completely sequenced.
A decade ago, a discovery was found that injection of double stranded RNA (dsRNA) into worms leads to
specific degradation of the corresponding mRNA - the process of RNA. Soon afterwards, it was found
that either soaking worms in dsRNA solution or feeding worms bacteria engineered to produce dsRNA
also could induce a robust RNAi response. The technique of RNAi, coupled with the availability of the
complete genomic sequence of C. elegans, has made possible the rapid study of gene function, both on
a single gene level and at a global scale.
Three techniques are being used for carrying out RNAi in C.elegans: by injection, soaking and by feeding;
all three can produce efficient gene knockdowns.
RNAi by injection: dsRNA produced in vitro is injected into young adult hermaphrodites and the progeny
scored for mutant phenotypes. RNAi by injection gives very reliable gene inhibition from worm to worm,
but is more labor intensive than other methods
RNAi by soaking: worms are soaked in a high concentration dsRNA solution and then subsequently they
or their progeny scored for phenotypes. RNAi by soaking is useful for treating a moderately large
number of animals or for high throughput screening in 96 well format. Worms of any stage can be
soaked. More dsRNA is needed for soaking than for injection.
RNAi by feeding: bacteria producing the desired dsRNA are fed to worms and either they or their
progeny are scored. RNAi by feeding is the least labor intensive and most inexpensive method, but
produces slightly more variable results than RNAi by soaking or injection. RNAi by feeding can be used to
treat a large number of animals at once or for high throughput screening, both on agar plates and in
liquid culture. Worms of any stage can be subjected to RNAi by feeding.





3. TILLING
Targeting Induced Local Lesions in Genome is a method in molecular biology that allows directed
identification of mutations in a specific gene. TILLING was introduced in 2000, using the model plant
Arabidopsis thaliana. TILLING has since been used as a reverse genetics method in other organisms such
as zebrafish, corn, wheat, rice, soybean, tomato and lettuce.
This technique involves mutagenesis with chemical mutagen (EMS, Ethyl methanesulfonate) or ENU
(ethylnitrosourea) and a sensitive DNA screening technique that identifies single base mutations called
point mutations in a target gene. EMS is a chemical mutagen that alkylates guanine bases. The alkylated
guanine will then pair with thymine instead of the preferred cytosine base, ultimately resulting in a G/C
to A/T transition. EMS is the most commonly used mutangen in plants. In Arabidopsis, five percent of
EMS-induced mutations in targeted coding regions result in premature termination of the gene product,
while fifty percent result in missense mutations that alter the amino-acid sequence of the encoded
protein. ENU also induces point mutations, and is a more potent mutagen than EMS. It is also an
alkylating agent, mutagenizing by transferring an ethyl group to oxygen or nitrogen radicals in the DNA
molecule, which leads to mispairing and ultimately results in base pair substitutions, and sometimes
base pair losses if not repaired.
The TILLING method relies on the formation of DNA heteroduplexes that are formed when multiple
alleles are amplified by PCR and are then heated and slowly cooled. A bubble forms at the mismatch
of the two DNA strands, which is then cleaved by single stranded nucleases. The products are then
separated by size on several different platforms.

TILLING Phytophthora
Zoospores present an ideal life stage for mutagenesis as they are uni-nucleate and single mutant
individuals can readily be isolated following mutagenesis. The mutation rate for EMS or ENU is not
known for Phytophthora and lethality is being used as an indicator of dose response. Genomic DNA is
extracted from the mutant individuals and pooled 2 to 4-fold. The genomic DNA library can then be
repeatedly screened. Specific genes are amplified from the pools of genomic DNA, and the PCR products
are heated up and allowed to cool slowly to form heteroduplexes between wild type and mutant strands
of DNA. The heteroduplexes are treated with the single strand specific endonuclease CEL1 which cuts 3
of single base mismatches producing novel fragments of DNA. CEL1 treated PCR products are then
resolved on a polyacrylamide gel and screened for the presence of novel fragments. Pools containing
novel fragments are then analyzed to determine exactly which mutant is carrying an induced point
mutation, and the PCR product from this mutant is sequenced to determine if the induced change is
predicted to be silent, missense, or a knockout mutation.
Since Phytophthora is diploid through most of its life cycle, including the zoospore stage, all of the
induced point mutations exist in the heterozygous state. Once a non-silent point mutation has been
identified the mutant isolate is taken through the sexual stage and the sexual progeny are screened to
identify individuals homozygous for the mutation under investigation. Homozygous mutants are then
tested to determine if there is an altered phenotype.

2. Procedure of TILLING Phytophthora


4. Gene disruption by homologues recombination
Homologous recombination is a type of genetic recombination in which nucleotide sequences are
exchanged between two similar or identical molecules of DNA. It is most widely used by cells to
accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks.
Homologous recombination also produces new combinations of DNA sequences during meiosis, the
process by which eukaryotes make gamete cells, like sperm and egg cells in animals. These new
combinations of DNA represent genetic variation in offspring, which in turn enables populations to
adapt during the course of evolution. Homologous recombination is also used in horizontal gene transfer
to exchange genetic material between different strains and species of bacteria and viruses.
Homologues recombination can be applied to gene targeting, protein engineering and cancer therapy
technological processes.

Gene targeting
Gene targeting by homologous recombination is associated with the repair of double strand breaks.
Many methods for introducing DNA sequences into organisms to create recombinant DNA and
genetically modified organisms use the process of homologous recombination.
Gene targeting is especially common in yeast and mouse genetics. In knockout mice it uses mouse
embryonic stem cells to deliver artificial genetic material which represses the target gene of the mouse
by the principle of homologous recombination. The mouse thereby acts as a working model to
understand the effects of a specific mammalian gene.
Advances in gene targeting technologies which hijack the homologous recombination mechanics of cells
are now leading to the development of a new wave of more accurate, isogenic human disease models.
These engineered human cell models are thought to more accurately reflect the genetics of human
diseases than their mouse model predecessors. This is largely because mutations of interest are
introduced into endogenous genes, just as they occur in the real patients, and because they are based
on human genomes rather than rat genomes. Furthermore, certain technologies enable the knock-in of
a particular mutation rather than just knock-outs associated with older gene targeting technologies.

Protein engineering
Protein engineering is a multidisciplinary technology for the design and construction of proteins. It offers
new possibilities for modification of natural proteins for specific industrial, clinical or agricultural
purposes, for combining proteins of two different cultures to reveal a new one, or for construction of
completely new protein which is not yet found in the evolution of the living organisms. There are three
approaches or strategies for protein engineering:
- De novo design (Knowledge-based protein design, achieved by modeling from homologues and
analogues proteins if available)
- Rational design (Here we use detailed knowledge of the structure and function of the protein
of interest to make changes. It can be done with site-directed mutagenesis techniques, but the thing is
that often we do not have the needed information for particular protein and if we do have it, sometimes
is hard to predict the effects of the mutations.)
- Directed evolution (In directed evolution, random mutagenesis is applied to a protein, and a
selection regime is used to pick out variants that have the desired qualities. This process mimics the
natural way of evolution. )

Cancer therapy
Cancer cells with BRCA mutations have deficiencies in homologous recombination, and drugs to exploit
those deficiencies have been developed and used successfully in clinical trials. Olaparib, a PARP1
inhibitor, shrunk or stopped the growth of tumors from breast, ovarian and prostate cancers caused by
mutations in the BRCA1 or BRCA2 genes, which are necessary for HR. When BRCA1 or BRCA2 is absent,
other types of DNA repair mechanisms must compensate for the deficiency of HR, such as base-excision
repair (BER) for stalled replication forks or non-homologous end joining (NHEJ) for double strand breaks.
By inhibiting BER in an HR-deficient cell, olaparib applies the concept of synthetic lethality to specifically
target cancer cells. While PARP1 inhibitors represent a novel approach to cancer therapy, researchers
have cautioned that they may prove insufficient for treating late-stage metastatic cancers. Cancer cells
can become resistant to a PARP1 inhibitor if they undergo deletions of mutations in BRCA2, undermining
the drug's synthetic lethality by restoring cancer cells' ability to repair DNA by HR.
Conclusion
Reverse genetics, like the classical one, is not ideal and not all techniques can be applied to all
organisms. Different approaches constitute of different costs and obstacles. Many scientists tend to
reveal what they seek and what they find, but not always that information is used in the right way. Even
if it is, there is no guarantee that things might get out of control. Trying to cure some disease may
results with emergence of some new type of disease, or creating a new product or even organism may
not give the desired outcome, but rather creating something totally new and therefore dangerous in
some way.
Many things were and still are questioned whether we should play or not with what we are given the
Mother Nature. Some things are better to be buried deep down than used by insane minds, but maybe
it is too late now.