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SUBMITTED TO : MISS MARYAM
SUBMITTED BY : GROUP 5 & 6
ASSIGNMENT TOPIC : TRANSGENESIS
OUTLINE:
 Introduction
 Transgenic vectors
 Methods and procedure of forgein gene preparation
 Methods of transgenesis
 Transgenesis markers and screening
 Application of CRISPR-CAS system
 Ethical point of view

TRANGENESIS
Introduction
Definition:
Transgenesis refers to the process of introducing transgene (i
...
an exogenous gene) from
one organism into another with the intent of enabling the latter to exhibit a new property that can
be transmitted to its offspring
...

One of the uses of transgenesis is to transfer genes (e
...
human genes) into animals
or cells to incite and observe human diseases
...
Transgenic mice are one of the most common animal
models used
...

These transgenic organisms are capable of expressing transgenes because the genetic
code is fundamentally similar for all organisms
...

The foreign gene of interest is prepared by different gene editing techniques like
recombinant DNA Technology, transcription activator-like effector nucleases (TALENs),
Zinc-finger nucleases (ZFNs), and CRISPR/cas9 (clustered regularly interspaced short
palindromic repeats)
...

The gene with the vectors is inserted to the host cell through different gene insertion
methods like heat shock,electroporation, viruses, gene gun, micro injection, and
liposomes
...

To date, the most ideal transgenicmarker is green fluorescent protein
...


Figure 1 Transgenic animals

Why use transgenesis instead of selective breeding?
• More specific — scientists can choose with greater accuracy the trait they want to
establish
...

• Faster — establishing the trait takes only one generation compared with the many
generations often needed for traditional selective breeding, where much is left to chance
...

• Less costly — much of the cost and labour involved in administering feed supplements
and chemical treatments to animals and crops could be avoided
...
Vectors increase the probability of gene
expression
...
Plasmid vectors
They are naturally occurring, small, self-replicating, circular and extrachromosomal pieces of
DNA isolated from bacterial cells
...

pUC18 is the most commonly used plasmid vector and they are restricted to accept DNA with
≤5000 base pairs
...
Specially developed bacteriophage
They have a characteristics future with one third of its genome is nonfunctional which made
them suitable for gene transfer
...
coli and can
integrate up to 15–16 kilobases of the DNA segment
...
Cosmids
They have both features of plasmids and bacteriophage chromosome without phage DNA so that
they reproduce as plasmids and can integrate DNA segments less than 50 kilobases
...
Yeast artificial chromosome (YAC)
It is a specially constructed linear yeast chromosome to incorporate less than 1 million base pairs
of DNA strands
...
It is done by using
conventional recombinant DNA techniques which functions by cutting and splice together pieces
of DNA resulting in recombinant DNA
...

Typical transgenes contain nucleotide sequences of the gene of interest with all components
essential for efficient expression especially the promoter or regulatory region such as that of
b-actin or simian virus 40T antigens and tissue-specific promoters
...
The major method for preparation of foreign gene of
interest is recombinant DNA technology which consists of the following three steps
...

Isolating the gene of interest
It is done by using recombinant DNA technology by using restriction enzymes
...

ii
...
The
vector containing a cloned gene is referred as chimera, which is introduced to the host cell to live
in different ways
...
Vector and
target DNA sticky ends should be produced by the same enzyme to be complementary to insert
the cut DNA in to the vector and joined by DNA ligase enzyme which covalently joins the sugarphosphate backbone of bases together
...

iii
...

a) Heat shock
Sudden heating of a solution containing cold calcium chloride with chimera plasmids and normal
host bacteria at 42°C for 2–5 min increases the permeability of the host bacterial membranes for
plasmid chimeras to be incorporated in the host cell
...

c) Viruses
The ability of viruses to infect susceptible cell and replicate themselves made possible the
incorporation of desired DNA sequence into the target host cells
...

e) Micro injection
By using a fine needle foreign DNA is injected directly into the nucleus by holding a cell place
with a pipette under a microscope
...


Methods of transgenesis
Efficient methods for incorporation of foreign DNA should exclude chemical or physical
mutagenesis
...
Genetically modified animals can be

totally altered or partially altered
...

1
...
Testis-derived cells transplanted into the testis of infertile males populate the host
testis, generate sperm, and produce offspring
...
Sperm-mediated gene transfer
It is based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA
molecules and to transfer them into the oocyte at fertilization used as alternative for micro
injection
...

3
...
It uses glass needle (i
...
, a fine, glass microcapillary pipette),
a precision positioning device (a micromanipulator) to control the movement of the micropipette,
and a micro-injector
...
It is the only successful method for producing GM livestock until recently with only 3% to
5% injected embryos get the transgene
...
It results in random incorporation of desiredgenome in the injected
embryos
...
It could not work for poultry because of
extreme difficulty to gain access to the fertilized egg when it is still at the single-cell stage
...
They have
the ability to integrate into the host DNA and copied when the cell divides
...
The pure homozygous
transgenic animals may result after 10 to 20 generations through inbreeding
and can be stored for subsequent implantation
...

Examples of viruses used for this purpose include Moloney Leukaemia Virus (causes lymphoid
leukemia in mice, rats, and hamsters); Rous Sarcoma Virus (cancer formation in humans) and
Avian Leukosis Virus (poultry flocks)
...

 Stem cell-mediated gene transfer:
According to Gordon (1996), Markkula and Huhtaniemi (1996) and Miao (2012), it involves
insertion of the desired gene into totipotent stem cells and the stem cells containing the gene of
interest are incorporated to the host embryos resulting in chimeric animals
...
It allows gene targeting by allowing directed modification of endogenous genes
 Somatic cell nuclear transfer:
The technique involves transfer of somatic cell nucleus to the cytoplasm of enucleated egg to be
reprogrammed by egg cytoplasmic factors to form a zygote
...

It starts during the mid-1980s, after 30 years of initial successful experiments with frogs and
now it is practicable in different species of animals except humans
...


Transgenic markers and screening of transgenesis
To test whether the cells incorporate the transgene or not, we should also incorporate transgenic
markers which should be screened in different ways
...

β-galactosidase, firefly luciferase, secreted placental alkaline phosphatase and green fluorescent
protein (GFP) are currently available transgenic markers but GFP is the most ideal marker
which allows selection of transgenic embryos soon after gene transfer or prior to embryo
transfer
...

The Southern blot assay is the most widely used for testing the presence of transgene in the
host animals
...
The fragments of DNA moved towards
the positive pole from the negative one and settled in the gel according to their size with the
bigger fragments on the top while the smaller fragments move faster to the gel and settled on the
positive pole
...
If the gene of interest is present
the blotted membrane picks the probe and illuminates the gene
...
SDS-polyacrylamide gel is used for electrophoresis
...
Then, it is incubated with
a primary antibody, which sticks to a transgenic protein to form protein-antibody complex and
visualization is made by hybridizing with the secondary antibody that forms a color
...

Enzyme-Linked Immuno-absorbent Assay is also used to identify the presence of transgenic
proteins by measuring the number proteins in serum, blood, and urine
...
If the sample contains transgenic
protein it reacts with the antigen and color is produced
...


Application of CRISPR-Cas systems in animal transgenesis
CRISPR-Cas adaptive immune system is formed by conjugation of Clustered regularly
interspaced short palindromic repeats (CRISPR) and associated sequences (Cas genes and Cas
proteins)
...
It consists cassettes of regularly alternating repeats and spacers, a leader
sequence and a set of Cas genes, which participate in different stages of the CRISPR-Cas
pathway to generate CRISPRRNAs (crRNAs) and Cas proteins
...


Animal Welfare And Ethical Concerns
The genetic modification of animals faced active opposition from animal welfare and ethics
point of views
...

Breaching the species barrier or playing God, which may result in unwanted characters (altered
behavior like enhanced aggression) and unknown hazards for humans is the major area of
opposition from religious peoples and animal right and welfare groups
...




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