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 cells
to 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 growth
hormone 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 DNA
microinjected 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 into
fish are human or rat growth hormone, chicken delta crystalline protein. E.coli β-
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 cell’s 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 inserted
into 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. It
also 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 remove
expression 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 electric
current. 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 life
cycle (this is described at http://www.ejbiotechnology.info/content/vol1/issue3/full/1/).
The mechanisms of this movement will be discussed in class. However, it is possible to
manipulate the plasmid such that a gene of interest is placed into the plasmid in
Agrobacterium so that the bacteria will introduce this DNA into a plant cell.

To transform most plants using Agrobacterium, a single plant cell that has received the
new DNA from the bacteria has to be regenerated into a whole plant. This process
involves the culturing of the transformed cell to provide replication of that cell. Levels of
plant hormones can be manipulated to cause this mass of cells to form roots and shoots of
a regenerated plant.This process can take weeks and the details vary from plant to plant.
For Arabidospsis, an alternate method has been developed- called dip infiltration. In this
method, Agrobacterium carrying the modified plasmid is introduced into the whole plant
by submerging the plant in a bacterial solution. Applying a vacuum can help force the
bacterial solution into the inner air spaces between plant cells, but this was found to be
not necessary. Agrobacterium will move the DNA from its plasmid into many of these
cells in the plant. Some of these transformed cells will be used to make the flowers of the
plant, including the pollen and ovules. With the self-fertilization possible in Arabidodpis,
seeds produced from these flowers will have the introduced gene at a low rate.

The major technical problem of transformation, regardless of the method used, is the low
frequency at which it occurs. Only a small fraction of cells where the DNA has entered
the nucleus does the DNA get spliced into a chromosome. Thus, one needs a way of
identifying those cells or plants that contain the introduce DNA. Usually, one gene
included in the introduced DNA is a selectable marker gene - for example a gene that
confers resistance against a chemical that kills normal plant cells (antibiotic inbroader
sense). Kanamycin is one such antibiotic that kills plant cells. Including a kanamycin
resistance gene along with a gene of interest in the Agrobacterium vector allows one to
select transformed plants by growing them on kanamycin. Only transformed plants will
survive since they express the introduced kanamycin resistance gene. For the
Agrobacterium infiltration method, the seed from the infiltrated plants are plated on agar
containing kanamycin – the low number of plants containing the introduced DNA will
germinate and grow on these plates.

We will use transformation to determine the function of genes through reverse genetics.
The cloned genes we will use are members of moderate to large gene families in
Arabidopsis. The encoded sequence of the genes clearly suggest the molecular function
by the presence of conserved protein motifs. One gene is a cellulase that digests the
cellulose in the cell walls of plants. Arabidopsis has more than 12 cellulase genes.
Another three genes we will study are myb DNA binding proteins. Arabidopsis has 125
myb genes that are expected to act as transcription factors. A fifth gene we will study is a
homeodomain protein also expected to be a DNA-binding transcription factor. Although
the biochemical activity can be predicted from the sequence, the function of these genes
would not be predictable from the sequence. To determine the process in which these
genes function, we will transform modified versions of theses genes into Arabidopsis and
determine how the phenotype of the plant changes. This change in phenotype can then be
linked to the function of the genes.
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 organs
of 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 or
environmental conditions, in response to internal or external cues. Since the expression of
genes is often regulated by transcription, the promoter (section of DNA preceding the
coding 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 all
times, 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 that
the 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 use
hybridization 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 organ
resolution 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 colorless
substrate 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 the
colored product . The β -galactosidase (lacZ) and β -glucuronidase (GUS) genes are two
examples 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 a
method 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 in
which 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 doesn’t know
if the β -galactosidase staining if from the introduced gene or the native plant gene. In
contrast, 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