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Applications of Microinjection Technique
Different applications of microinjection technique in plant
science have been reported.
Some experiments deal with the introduction of fluorescent dyes
into differentiated cellsto study intracellular transport. Efforts
have been made to microinject microorganisms
into suspension cells. Attempts to transfer chromosomes or even
cell organelles have
been made. A common feature of these experiments is low numbers
of manipulated cells.
DNA is microinjected in fish. The injected zygotes are cultured
in vitro to blastocysts and
then placed inside a foster mother. This technique has been used
to introduce rat growthhormone gene. Microinjection of foreign DNA
into newly fertilized eggs was first
developed for the production early 1980s. Since 1985, this
technique has been adopted
introducing trans genes into Atlantic salmon, common carp,
catfish, goldfish, loach, and
zebra fish. As the male pronucleus is difficult to locate in
fish zygote, the DNAmicroinjected into cytoplasm of fertilized
ovule within first few hours after fertilization.
As the Fishes undergo external fertilization, transfer of
embryos into foster mothers is not
needed. Transformed embryos are instead hatched in trays.
Various gene transferred intofish are human or rat growth hormone,
chicken delta crystalline protein. E.coli -galactosidase gene,
E.coli neomycinresistance gene and E. coli hygromycin
resistance
gene. Gytoplasmic microjection is a tedious and time-condsuming
procedure and is
unsuitable for mass transfer.
Genetic transformation
Transformation is the introduction of DNA representing a cloned
gene into a cell so that
it expresses the protein encoded by the gene. Although the
physical insertion of DNA
into a cells nucleus is straightforward, the expression of
proteins encoded by that DNA
that is not part of a chromosome is often only transient.
Introduced DNA that is insertedinto one of the chromosomes will be
passed during mitosis to all subsequent daughter
cells. It is this stable transformation that will allow one to
introduce one copy of DNA
into one cell, and then allow the one transformed cell to
regenerate a complete organism,where each cell contains a copy of
that introduced DNA. The manipulation of an
organisms DNA by transformation allows unparalleled ability to
determine the function
of a gene from levels of cell function, to organismal physiology
to ecological roles. Italso provides a way to dissect the
functional significance of parts of the gene or specific
amino acid residues of the resulting protein. Transformation
additionally allows the
engineering of plants or animals to produce novel proteins or
specifically removeexpression of certain proteins.
There are a number of ways that cloned DNA can be physically
introduced into a cell.
DNA can be micro-injected into cells, or shot into the cell on
the surface of
microprojectiles, or enter through holes in the cell membrane
induced by a strong electriccurrent. Drosophila and C. elegans are
usually transformed through microinjection. Plant
transformation can take advantage of a plant pathogenic bacteria
(Agrobacterium
tumifaciens) that move DNA from a plasmid it carries into plant
cells as part of its lifecycle (this is described at
http://www.ejbiotechnology.info/content/vol1/issue3/full/1/).
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Reporter genes
Arabidopsis contains nearly 26,000 genes. Some of these genes
are expressed at most
times in every cell. However, the majority of genes are only
expressed in certain organsof the plant, either causing that organ
to be different than other organs, or adding function
to that organ. Further, many genes are only expressed under
certain developmental orenvironmental conditions, in response to
internal or external cues. Since the expression of
genes is often regulated by transcription, the promoter (section
of DNA preceding thecoding region), will contain the information
that allows the gene to be turned on or off in
different organs or in response to cues. The Cauliflower mosaic
virus (CaMV)35S
promoter is one of the few plant promoters that is expressed in
most every tissue at alltimes, called constitutive.
An important clue to the function of a gene is to determine
where and when it is
expressed. If it is expressed only in flower stamens, then it is
apparent that it has some
role in male gamete formation or stamen development. If it is
only expressed under
certain conditions, such as after exposure to damaging UV light,
it would be apparent thatthe gene has a role in responding to such
stress or repair. There are several ways of
determining where and when a particular gene is expressed in a
plant. One way is to usehybridization to detect the amount of mRNA
corresponding to a cloned gene in samples
from different parts of a plant, sampled after different
treatments of the plant. This
approach is quantitative but is time-consuming and provides only
as much time or organresolution as the researcher has patience for
separating different parts of many plants to
gather sufficient quantities of mRNA samples. Another approach
is the use a reporter
gene. A reporter gene produces a protein that is easily
detectable in transformed
organisms. Often, the protein possesses an enzymatic activity
that can turn a colorlesssubstrate into a colored product. Thus,
one can see the location and amount of gene
expression in a transformed organism by looking at the location
and intensity of thecolored product . The -galactosidase (lacZ) and
-glucuronidase (GUS) genes are twoexamples of these reporter genes.
When the reporter gene is fused to the promoter of the
gene of interest, the reporter gene will be expressed only at
the times and locations where
that gene is expressed since the promoter often determines
transcription. This provides amethod to detect a very limited
expression of a gene, such as in small patches of cells
(like root tips or pollen) or at certain times (such as after a
certain stress or hormone
treatment).
An important property of reporter genes is that their activity
is absent in the organism inwhich they will be used. Both lacZ and
GUS are genes from E. coli. Plants posses some
LacZ activity, and so it is difficult to use it as a reporter
gene because one doesnt knowif the -galactosidase staining if from
the introduced gene or the native plant gene. Incontrast, GUS
activity is normally very low in plants, and so is a common
reporter gene
used in plant studies. Genes we will use promoters from for GUS
fusions are:
cellulase -AT1G64390
Myb60
