From Wikipedia,
the free encyclopedia.
Reverse genetics is an
approach to discovering the
function of a
gene that proceeds in the
opposite direction of so called
forward genetic screens that
are more usual in
classical genetics.
Classical and reverse genetics
are alike since using either
approach investigators try to
deduce information from the
effects of damaging or changing a
genes function. By the classical
approach, geneticists first look
for rare individuals with an
unusual
phenotype and then attempt to
identify the unknown faulty
allele or gene. In contrast,
for reverse genetics, the goal is
to identifying a known gene's
phenotype.
Due to the modern techniques of
DNA sequencing vast amounts of
genomic sequence data
available and many genetic
sequences are discovered in
advance of other information. To
learn the influence a sequence has
on phenotype, or to discover its
biological function, researchers
can engineer a change or
disruption in the DNA. After this
change has been made a researcher
can look for the effect of such
alterations in the whole organism.
There are several different
approaches to identify new alleles
in a known gene.
Random deletions, insertions
and point mutations
These are three similar
techniques that involve creating
large mutagenised populations in a
similar way to forward
genetic screens. These
populations are generated using
either chemical (point mutations),
gamma radiation (deletions) or DNA
insertions (insertional
knockouts). These large libraries
of mutants can be screened for
specific changes at the gene of
interest using
PCR. For some organisms, such
as
Drosophila and
Arabidopsis there are large
online databases that indicate the
locations of all the DNA
insertions in a particular
library.
Directed deletions and point
mutations
Site-directed mutagenesis is a
sophisticated technique that can
either change regulatory regions
in the
promoter of a gene or make
subtle
codon changes in the
open reading frame to identify
important amino residues for
protein function.
Alternatively, the technique
can be used to create
null alleles so that the gene
is not functional. For example,
deletion of a gene by
gene knockout can be done
in some organisms, such as yeast
and
mice. In the case of the yeast
model system directed deletions
have been created in every
non-essential gene in the yeast
genome.
In some cases conditional
alleles can be used that have
normal function until the allele
is activated. This is known as
gene knockin. This might
entail ‘knocking in’
recombinase sites (such as lox
or frt sites) that will cause a
deletion at the gene of interest
when a specfic recombinase (such
as CRE, FLP) is induced. Cre or
Flp recombinases can be induced
with chemical treatments, heat
shock treatments or be restricted
to a specific subset of tissues.
Gene silencing
The discovery of gene silencing
using double stranded RNA, also
known as
RNA interference (RNAi), and
the development of gene knockdown
using
Morpholino oligos have made
disrupting gene expression an
accessible technique for many more
investigators. This method is
often refered to as a
gene knockdown since the
phenotype is rarely due to a
complete loss of function.
RNAi creates a specific
knockout effect without actually
mutating the DNA of interest. In
C. elegans, RNAi has been
used to systematically interfere
with the expression of most genes
in the genome. RNAi acts by
directing cellular systems to
degrade target messenger RNA
(mRNA).
While
RNA interference relies on
systems within the cell for
efficacy (e.g. the dicer proteins,
the RISC complex) a simple
alternative for gene knockdown is
Morpholino antisense oligos.
Morpholinos bind and block access
to the target mRNA without
requiring the activity of cellular
proteins and without necessarily
accelerating mRNA degradation.
Morpholinos are effective is
systems ranging in complexity from
cell-free translation in a test
tube to humans.
Interference using transgenes
A
molecular genetic approach is
the creation of
transgenic organisms that
overexpress a normal gene of
interest. The resulting phenotype
may reflect the normal function of
the gene.
Alternatively it is possible to
overexpress mutant forms of a gene
that interfere with the normal (wildtype)
genes function. For example, over
expression of a mutant gene may
result in high levels of a
non-functional protein resulting
in a
dominant negative interaction
with the wildtype protein. In this
case the mutant version will out
compete for the wildtype proteins
partners resulting in a mutant
phenotype.
Other mutant forms can result
in a protein that is abnormally
regulated and constitutively
active (‘on’ all the time). This
might be due to removing a
regulatory domain or mutating a
specific amino residue that is
reversibly modified (by
phosphorylation
methylation or
ubiquitination). Either change
is critical for modulating protein
function and often result in
informative phenotypes.
