1.

identification of the gene interest;

growth hormone (GH) - enhanced growth is associated with more
effective utilization of food
Type iii anti-freeze proteins (AFP) - reduce the freezing temperature by
preventing ice-crystal growth ( cold winter can severely damage both
production and brood fish stocks of fish.)
infectious hematopoeitic necrovirus (IHNV) glycoprotein under the
control of the cytomegalovirus promoter (pCMV)- increase the resistance
of fish to pathogens.
2. isolation of the gene of interest;
ocean Pout Anti-Freeze Protein (opAFP): a genomic isolate of a Type III
anti-freeze protein (AFP) gene from the ocean pout (op)
Chinook Salmon Growth Hormone (GH): The Chinook salmon GH gene
was identified and isolated from a cDNA library prepared using pituitary
gland of Chinook salmon. This cDNA is full-length and encodes a single,
mature hormone
3. cloning the gene of interest
Polymerase Chain Reaction (PCR) enables the gene to be multiplied to the level
of several million copies needed for the generation of the construct.
gene of interest has been ligated enzymatically into the construct, this whole
complex is ligated into bacterial plasmids. which act as production vectors
and enable the gene to be replicated many times within the bacterial cells
4. the construct
A construct is a piece of DNA which functions as the vehicle or vector carrying
the target gene into the recipient organism. There is a promoter region which
controls the activity of the target gene, a region where the target DNA is
inserted, usually some type of reporter gene to enable one to ascertain whether
the target has combined successfully with the construct and a termination
sequence

5. techniques for inducing transgenic

DNA microinjection This method involves the direct microinjection of a
chosen gene construct into a fertilized ovum The insertion of DNA is,
however, a random process, and there is a high probability that the introduced
gene will not insert itself into a site on the host DNA that will permit its
expression

production plan
Female homozygous are used as the source of gm salmon
Eggs from qualified homozygous females are then subjected to
gynogenesis, resulting in an all-female gynogen offspring population
Fish eggs will only undergone the first meiotic division and leaves the
eggs with 2n number of chromosomes because when the sperm enters the
egg, meiosis 2 occurs will destroy the genetic material
The eggs are subjected to hydrostatic pressure shocks at the correct time
after the sperm enters the egg, to prevent meiosis 2

 Therefore, there is no genetic contribution from the male, and there are
two sets of chromosomes contributed by the female
all-female gynogen population is then masculinized through the
application of 17-methyltestosterone, so that they produce milt (fish
semen) instead of viable eggs
Collection of the milt from neomale broodstock is a terminal procedure,
as these fish lack seminal vesicles for normal spawning
Each neomale may only used for one production cycle , can fertilize
approximately 50,000 eggs
Broodstock salmon (neomales), upon sexual maturity, are then outcrossed with non-GE Atlantic salmon females
Fertilized eggs are then subjected to pressure shock treatment in order to
render the offspring triploid, with two copies of the genome coming from
the non-GE female and one copy from the homozygous neomale gynogen
This is the same procedure as with the development of gynogens, except
that this time the sperm nuclei contributes 1n number of chromosomes,
and the only sex chromosome it can contribute is a third X chromosome
Female triploid salmon are effectively reproductively incompetent,
providing additional environmental and intellectual property safeguards.
the eggs are then subjected to pressure shock treatment, causing them
to produce diploid offspring (to prevent meiosis 2.Therefore, there is
no genetic contribution from the male.) and there are two sets of
chromosomes contributed by the female. The all-female GE diploid
salmon will then be treated with 17-methyltestosterone, a hormone
that turns the fish into neomales genetically female fish that
produce milt (sperm) instead of eggs. The milt from the GE
neomales will fertilize the eggs of non-GE Atlantic salmon, and the
resulting fertilized eggs will be treated with pressure to produce the
final product, a line of all-female triploid GE Atlantic salmon.

chinook
contain growth hormone
isolate this gene clone it in bac and read its sequence of genetic code
both genetic code is similar in the two fish
eel like fish live in very cold waters
contain promoter turn on a gene that makes an sntifreeze chemical
in gmo salmon the same promoter has a different job: keeping the growth
hormone gene turned on all the time. Atlantic salmon the gene is switched on
only during summer months