GENETICALLY MODIFIED ORGANISMS

❖ Definition

According to the European Union (EU), genetically modified organisms (GMOs) are defined as
any organism, except humans, carrying an altered genetic material that does not occur
naturally through natural selection or mating in the Directive 2001/18/EC on the deliberate
release into the environment of GMOs issued by the EU and including techniques of
recombinant DNA (rDNA) technology along with microinjection techniques as a tool for genetic
modifications. All such tinkered organisms are known by synonymous terms like “genetically
engineered,” “genetically modified, “transgenic,” “biologically engineered,” and “biologically
modified” organisms.

❖ Advent of GMOs

A scientific breakthrough was achieved around three decades ago when the first recombinant
mouse was generated through microinjection into the pronuclei of mouse oocyte. Since then,
genetically modified (GM) mice have been an indispensable part of medical research, serving
as disease models for various congenital, metabolic disease, cancer, and aging disorders. Other
animals have also been genetically modified for reasons other than research. Considerable
success has been achieved in the introduction of novel traits into different animals. There is
now an extended list of cows, swine, sheep, and goats genetically modified or fortified for food
(and other applications) generated through various rDNA technologies and epigenetic
approaches (reviewed later in the chapter), creating heritable and non-heritable changes in the
genome. Transgenesis is an extension of the conventional breeding practices:

● Being more robust, direct in approach, and effective than conventional breeding;

● Bringing about heritable changes once a foreign gene is inserted;

● Not restricted by fertility barriers in which only closely related species could be
crossed, even heterologous genes have been inserted into animals with substantial
success, for example, the EnviropigTM, containing a phytase-producing gene from
Escherichia coli; and
● Inducing both heritable and non-heritable changes with the usage of appropriate
methodology.

● Methods for Introduction of Transgenesis

The introduction of transgenesis can be roughly grouped into three categories based on the
changes in the genome desired:
1. The introduction of a new gene,
2. The replacement of a gene through homologous recombination, and
3. The alteration of gene expression through epigenetics.
Before venturing into the details of the methods the foremost point of consideration is
identification of the gene of interest.

❖ Identification of the Gene of Interest

Identification of the gene of interest is the most vital spoke in the wheel of genetic engineering.
When generating a GM organism, a transgene is inserted into the host animal’s genome.
Among the many available routes for screening and identification of the gene of interest, the
exact approach would depend on the organism and the rDNA technology available for that
organism, along with other factors like whether the mutation is recessive or dominant, which
screens are available for identification of the mutant and the wild-type phenotype, known
homology with other genes, etc. The gene of interest can be screened from sequence
homology, targeting the RFLP (restriction fragment length polymorphism) markers around the
gene of interest, or from the cDNA libraries (utilizing the technique of “primer walking”)
available in the public domain or by creating your own libraries.

I. Microinjection
Microinjection or pronuclear injection was at one time the most successful method for
producing GM animals. Around 400–500 copies of the transgene construct are directly
injected using a very fine needle into one of the two pronuclei of the fertilized embryo.
Transgene gets inserted into the host genome randomly with low efficiency and such in-vitro
fertilized embryo thus generated is then cultured in the artificial environment and transplanted
into an impregnated surrogate mother. Microinjection at a later stage (after the single celled
stage) leads to the appearance of “mosaic” patterns.
The microinjection technique has been used most widely for biopharming. This method has its
own lacunae, too, like a very low degree of efficiency of ∼5%, high screening costs, and being
unsuitable for creating GM birds/poultry as it is challenging to access the fertilized egg at the
single-celled stage. Therefore, the method of viral transfection is best suited in case of aves.
Nonetheless, microinjecting the gene construct into the zygote was reported to produce
transgenic birds with some success. Advances in combining pronuclear injection with
integrases and recombinases hold greater possibilities for creating targeted mutagenesis. The
first report of generating transgenic mice with high muscle mass through direct microinjection
of the recombinant lentiviral vector containing shRNA against the myostatin gene holds
promise for quality food from livestock.
 II. Viral Transfection
Lentiviruses—slow growing members of the viral family of retroviridae categorized by long
incubation periods—have been the most utilized vectors to deliver the transgene by virtue of
their ability to integrate with the host genomic DNA and proliferate with every replication of the
host genomic content. However, adenoviruses—the non-enveloped double stranded DNA
viruses of the family adenoviridae are also being used. Recombinant adenoviruses have been
used in swine to generate vaccines against swine influenza virus. The “replication competent”
viruses reintegrate the transgene multiple times and ensure transgene overexpression in the
offspring and sometimes even in other animals coming into contact, due to their high infection
rates. In contrast, “defective competent” viruses can transfect the cells only once.
Retrovirus-mediated transfer has been successfully used to transfer a foreign gene into cows,
chickens, monkeys, swine, and sheep through mammalian and avian retroviruses like Rous
sarcoma virus (RSV), Moloney leukemia virus (MLV), Avian leukosis virus (ALV), Simian virus,
etc. Viral- mediated genetic modification has some major practical limitations:
1. It limits the size of the transgene to be inserted, making it unviable for larger gene
constructs.
2. The long terminal repeats of the virus interfere with the promoter of the transgene.
3. Viral vectors lack the ability to replicate in early embryo cells, resulting in “chimeras”
where the transgene is expressed in only some of the cells of the GM animal (McCluskie
et al., 1999).
III. Embryonic Stem Cells Modification
Embryonic stem (ES) cells are self-renewing pluripotent cells derived from the inner cell mass
of early dividing embryos called blastocysts. The first successful establishment of the
embryonic stem cell culture was carried out from a murine embryo in the early 1980s and
concerted the effort for the isolation of ES cells from nonhuman primates and finally from
humans.
The transgene delivery system, as in the viral delivery system, could be “directed” or “random,”
depending on the strategy implied. Directed gene “knock-in” or “knock-out” relies on
homologous recombination, a DNA repair mechanism where the nucleotide sequences are
exchanged between genomic and exogenous sequences through the event of crossing over
between homologous sequences. The transformed ES cells are then transferred into a recipient
embryo, implanted into an impregnated surrogate, and “chimeras” or “completely
transformed” animals are screened. Unfortunately, this technology is still in its infancy as stable
ES cell lines have not been established for livestock.
IV. Spermatocyte-Mediated Gene Transfer
Transgene is incorporated into the spermatocyte through electroporation, polyethylenimine-
mediated gene transfection, or viral-mediated delivery. The transfected spermatocyte is used
as a vector after in-vitro fertilization, where the attachment of exogenous DNA to the sperm is
facilitated by the specific DNA binding proteins (DBPs) present on the post-acrosomal surface
of the sperm. The first transgenic mice were generated through sperm-mediated gene transfer
by Lavitrano et al. (1989), mixing plasmid DNA with spermatocytes. But the results of sperm-
mediated gene transfers are marred by low repeatability and efficiency in the case of
mammals. Intracytoplasmic sperm injection directly into unfertilized oocytes has substantially
increased the efficiency of transgene expression in the recovered offspring.
V. Cloning: Somatic Cell Nuclear Transfer (SCNT)
The emergence of cloning technologies facilitating nuclei transfer from the somatic cells
brought a paradigm shift in the biological science in generating transgenic livestock. In somatic
cell nuclear transfer, enucleation of oocytes is followed by the fusion of the donor cell and
activation of this reorganized embryo. SCNT has been efficacious in many animal species,
including those reared for domestic, wildlife, or laboratory research. It has been the method of
choice for generating larger transgenic animals, beginning with the cloning of Dolly (the first
cloned animal) and later carried out in other animals, producing transgenics with greater
muscle mass and therapeutic applications. It is a potent tool for the extension of superior
breeding stock of farm animals for food.
VI. Transposon-Mediated Gene Silencing
Transgenesis can be induced in livestock through transposons. Transposons, or “jumping
genes,” are the unique DNA elements that traffic around the genome. This inherent property of
these elements can be harnessed to induce genetic modification in genome generating
transgenic livestock. With the discovery of sleeping beauty, PiggyBac- and Tol2-like
transposons, interest has been reinvigorated in this domain. Successful generation of
transgenic swine has been achieved through the introduction of double stranded RNAi
(ribonucleic acid mediated interference) cassette silencing the Cystic fibrosis transmembrane
conductance regulator (CFTR) through the introduction of transposons (Sleeping
Beauty/Tol2/PiggyBac, etc.) flanking the nucleic acid construct containing the transcriptional
unit on both sides. Transgenic rabbits and pigs carrying a fluorescent reporter have also been
generated using a similar approach.

❖ APPLICATIONS OF TRANSGENIC ANIMALS

Genetically modified animals have found greater applications in medical research with many
animal models being generated for different diseases. However, due to lower costs, efficiency,
reliability, and reproducibility, biotechnology companies have invested in GM animals as well,
taking greater interest in the applications of GM animals for food, therapeutics, organ farming,
recreation, environmental sustainability, etc. Numerous commercialization applications are
filed every year with the regulating agencies.

1. Biological Research
Most GM animals that have been developed as of this writing fall under the category of
biological research, with the most number of transgenic rodents and to some extent rabbits,
guinea pigs, and pigs being developed to act as disease models for neuropsychiatric, hearing,
and vision research, as well as, many types of cancers, immunodeficiency, and cardiovascular
and metabolic disorders.
2. Xenotransplantation

Being evolutionarily close to humans, pigs are the most common animal species projected to be used in this regard. A r
 3. Biopharming

The commercial production and extraction of pharmaceuticals from body secretions of GM animals is called biopharm

4. Environmental Sustainability
The generation of EnviropigTM was a major step in making biotechnology more environmentally
sustainable through reducing its environmental footprint. EnviropigTM produce 70% less
phosphorus in their manure due to the introduction of the phytase enzyme from Escherchia
coli, which enables the pig to produce a plant phytic acid digesting enzyme in its salivary
glands, reducing the cost of mineral phosphate supplements.
5. Food
Farm animals are reared at a large scale for human consumption but the growing demand for
animal meat, milk, and other products is difficult to meet. AquAdvantageTM, a GM Atlantic
salmon (a product of AquaBounty Technologies in Maynard, Massachusetts), is all set to
become the first commercialized GM animal approved by the Food and Drug Administration
(FDA) for consumption. AquAdvantage contains a promoter sequence from ocean pout and the
growth hormone gene sequence from the Pacific Chinook salmon (Oncorhynchus
tshawytscha). AquAdvantage and Chinook salmon belong to different genera, ruling out the
possibility of natural inbreeding. It is slated to clear the 7-step FDA approval process.