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Hello. In the lecture, we will focus on the basics 
of food safety, primarily in connection with GMOs,

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and describe the essence and significance of 
GMOs and animal cloning. The lecture is part

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of Module 2: Conservation and Sustainable Use of 
Animal Genetic Resources. The creation of this

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presentation was supported by the ERASMUS+ 
KA2 grant within the ISAGREED project:

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Innovation of Content and Structure of Study 
Programs in the Field of Management of Animal

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Genetic and Food Resources Using Digitization.
Food safety is currently one of the most important

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topics in relation to animal husbandry and 
food production. From this perspective, it is

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necessary to monitor the potential occurrence 
of harmful chemicals that can have mutagenic

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or even carcinogenic properties. Here, we most 
commonly encounter the presence of heavy metals

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and mycotoxins. In some countries, antibiotics may 
also be present in meat as unwanted contamination.

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Food must have a defined composition to prevent 
the occurrence of naturally occurring allergens,

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and in this case, consumers need to be notified 
of this risk. The most serious in terms of health

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is microbiological contamination, especially 
in meat and dairy products, by bacteria such

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as salmonella, listeria, or Campylobacter. 
Fortunately, the presence of these foodborne

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pathogens can be tested using microbiological 
cultivation techniques, but also DNA tests.

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In connection with the presence of GMOs in food, 
thorough testing of their health safety is carried

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out during their approval. With GMOs, there is a 
potential risk of allergenicity, so even they need

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to be tested and detected. Similar mechanisms 
for verifying health safety need to be applied

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to potential foods made from cloned animals.
Currently, the presence of GMOs above 0.9% must

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be declared in food. For this purpose, several 
sensitive detection methods have been developed.

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The diagnosis of GMOs in food can be performed 
by detecting the presence of GMOs through

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direct identification of transgenic DNA (using 
polymerase chain reaction - PCR) or by detecting

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transgenic protein - immunochemically using the 
ELISA method. In the case of using PCR, if a

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transgene is present in the sample, amplification 
or replication of the product occurs, which is

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visible as a band on an electrophoretic gel.
Quantitative methods are used to determine the

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proportion of GMOs in food. The most 
sensitive method is real-time PCR,

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which is extremely accurate and sensitive. The 
amount of GMO in the sample is determined by

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comparing the curve of the tested sample with 
the curves of known composition standards. The

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method is also called relative quantification.
In terms of the result, there is no difference

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between traditional breeding and transgenesis as 
the main method of creating genetically modified

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organisms. They have the same goal, which is 
to obtain individuals with desirable alleles

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in their genotypes. In traditional breeding, 
hybridization and deliberate crossing are often

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used, and it takes many generations to obtain 
desired combinations of genotypes. Transgenesis

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is characterized by targeted and rapid changes, 
requiring knowledge of the gene we want to use

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and transferring it to the organism using 
a vector, i.e., its structure, function,

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and sequence. Traditional breeding work is done 
in the population, while transgenesis can be

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seen as breeding an individual. The comparison 
of both approaches can be seen in the diagram.

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First, let's define genetic modification. Genetic 
modifications refer to targeted interventions in

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genetic information. Although the result may 
be similar, random interventions by mutagens

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or ionizing radiation (such as the creation of 
wheat or rapeseed varieties) are not considered

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genetic modification. Genetic modifications 
include intentional changes in gene activity,

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changes in "site of action" (in which tissue), 
gene replacement with another variant,

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gene blocking, and especially the introduction 
of foreign genes - transgenesis. A classic

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example of transgenesis in plants is the use 
of the bacterium Agrobacterium tumefaciens,

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which has the natural ability to introduce 
genes for herbicide resistance or insecticide

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production - this is how Bt corn, for example, 
was created. Alongside the development of

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these biotechnologies, legal regulations are 
emerging, such as the law on handling GMOs.

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Genetic modifications are synonymous with 
recombinant DNA techniques. These involve

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direct and targeted interventions in the 
hereditary material of an organism (i.e.,

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DNA). The most well-known method is transgenesis, 
also known as gene transfer between species and

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the introduction of individual genes into the 
genome using genetic engineering methods. A

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genetically modified organism (GMO) is an organism 
(other than a human) whose genetic material has

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been intentionally altered in a manner that cannot 
be achieved through natural recombination. GMOs

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can be microorganisms, fungi, plants, or animals.
The creation of GMOs presents a few problems.

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One of them is the low efficiency of insert 
incorporation. The integration of an insert and

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its copies often occurs randomly. The product can 
be formed in low or high concentrations because it

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is difficult to properly regulate expression 
of the structural gene. Incorporation of

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foreign DNA is often unstable and may be lost over 
generations. Gene manipulations are still costly,

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and the goal is often achieved with great 
uncertainty. The most advanced molecular

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biotechnology based on the CRISPR-Cas9 
system helps solve most of these problems.

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The base of recombinant DNA technology 
(used in genetic engineering) lies in the

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use of restriction enzymes, which originate from 
bacteria and serve as a defense mechanism against

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foreign DNA. These enzymes can cleave DNA at 
specific sites known as restriction sites. By

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using the same enzyme to open the vector and 
create both ends of the gene, the likelihood

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of correct insertion of the gene into the plasmid 
can be increased. The DNA ligase enzyme is used

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for this joining process of originally unrelated 
DNA segments, and it is called DNA recombination.

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The vector can then be integrated into 
the genome of the host cell, thereby

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transferring the sequence of the gene itself.
The main problem with transgenesis is how to

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effectively introduce recombinant DNA into 
the cell and nucleus. Many methods have

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been developed, of which I will first mention the 
so-called biological methods. Lipofection involves

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the use of lipid micelles that encapsulate 
nucleic acids into liposomes, which can naturally

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penetrate the cell nucleus. Transfection using 
plasmid vectors is a relatively simple method

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but with low efficiency. Viral vectors are 
now more commonly used in animals because they

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have a natural ability to penetrate the cell and 
nucleus. In the case of adenoviruses, DNA enters

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the chromatin, not directly the DNA, while in the 
case of retroviruses and lentiviruses, foreign DNA

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directly integrates into the genome. The image 
on the right shows the schema of a lentivirus,

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which, in addition to RNA, contains enzymes for 
integrating nucleic acids into the host's DNA.

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During transgenesis, care is taken to ensure 
that viral vectors are safe and modified so

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that they cannot reproduce in the host cell.
Physical methods mainly involve microinjection,

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which is the introduction of DNA into a fertilized 
egg or embryonic stem cells. This method is simple

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and foreign genes are effectively expressed. 
The method cannot be used in later developmental

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stages, and the disadvantage is low success 
rate and random insertion of the insert. Gene

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transfer using embryonic stem cells, known as 
ESC, is a specialized method where pluripotent

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cells from a blastocyst with DNA inserted 
in vitro are inserted into a foreign embryo,

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which is then transferred to a surrogate mother's 
uterus. The result is the birth of an offspring

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that is a genetic chimera, meaning it has some 
tissues with the transgene and others without.

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Other methods include transfer of DNA through 
various particles and shooting them into the cell,

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as well as electroporation (i.e. creating pores in 
the cell using electrical impulses). Thermal shock

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or coated magnetic particles can also be used. 
However, all of these methods have relatively

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low efficiency (up to 5%) and their application 
depends on the specific species of animals and the

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experience of the respective laboratory.
In this scheme, we see an example of

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microinjection technique used to transfer 
a transgene, with the aim of expressing the

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transgene only in the mammary gland and producing 
a readily isolatable product, i.e. protein,

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from transgenic milk. These genetically 
modified animals used to produce specific

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transgenic proteins are called animal bioreactors.
Various uses of microinjection can be seen in the

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images here. Injection can be done into the 
pronucleus just before the fusion of nuclei

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and the formation of a zygote - the result is 
a completely transgenic individual. Another

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approach is the insertion of the transgene into 
embryonic stem cells, which are then inserted into

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the host blastocyst. Chimeras are born, which can 
be further crossed and in subsequent generations,

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a completely transgenic individual can be 
obtained again. The advantage of this method

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is that we can culture and select embryonic 
stem cells, which increases the likelihood of

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success in transgenesis. Another frequently 
used method is nuclear transfer, where a DNA

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construct is inserted into cultured somatic cells 
(usually undifferentiated, such as fibroblasts),

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and then the nucleus is removed and inserted 
into an enucleated egg. The zygote is then

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implanted into a surrogate mother and in all 
cells transgenic individual can be born again.

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And what is the purpose of creating GM animals? 
First, to increase the better production and

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quality of animal food. This is also related 
to the production of new and better foods,

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for example the lactase gene in cattle will reduce 
the lactose content in milk. Another example is

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the replacement of allergenic proteins in 
milk with human proteins. There is great

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potential for the production of high-quality 
recombinant proteins (pharmaceuticals,

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etc.) or new materials in industry with the help 
of so-called "living bioreactors". High hopes are

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placed in research on resistance to disease and 
the adverse effects of a changing environment,

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for example, an experiment with the transfer of a 
gene for a protein that protects against freezing

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into the genome of salmon. The use of animal 
models is important for research into human

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diseases, and research into the use of transgenic 
animals for xenotransplantation is also underway.

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The most significant application of genetically 
modified animals is in the field of pharmaceutical

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production. Many important drugs, often for 
the treatment of genetic diseases, are of

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protein nature. Proteins, as complex substances, 
cannot be produced chemically like simple drugs,

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but synthesis must occur in biological systems. 
The simplest way is the production of drugs using

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microorganisms, such as bacteria. Insulin has 
been produced this way for many years. However,

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insulin is a relatively simple protein, and 
bacteria cannot produce more complex proteins

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with specific post-translational modifications. 
Therefore, eukaryotes - animals such as mammals or

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birds - must be used. One of many examples of drug 
production is obtaining human protein lipoprotein

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lipase from chicken eggs. Goats or rabbits are 
often used for similar purposes in mammals.

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Proteins have very diverse properties that 
can be used as materials for various advanced

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technologies. For example, spider silk protein 
is a material that is about 7 times stronger

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than high-quality steel in terms of volume 
and weight, and it is called Biosteel. The

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source of this protein is genetically modified 
goats that produce this protein in their milk.

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There are not many examples of genetically 
modified food of animal origin approved for

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consumption. A well-known example is GM 
salmon, which grows 11 times faster than

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regular farmed salmon. The approval process 
in the United States was very complicated and

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took 20 years. To prevent the escape of GM salmon 
into the wild, they are bred in isolated tanks.

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Genetic modification is also associated 
with ethical problems. Is the new product

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acceptable to customers? European consumers 
tend to reject GMO foods. In the past,

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concerns arose regarding the risk of 
tumor formation or neurodegenerative

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diseases in transgenic animals due to the 
integration or expression of transgenes,

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following the initial failed experiments. It is 
logical that side effects due to gene modification

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cannot always be excluded. An ethical issue 
for many people may be that humans may benefit

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from genetically modified animals even if the 
transgenic animals themselves do not. In any case,

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it is necessary to ensure that GM does not 
cause any harm to animals. There are often

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concerns that foreign genes affect hosts and 
whether there may be a threat to ecological

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balance and species diversity. However, GM 
animal husbandry is usually closed, although

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there is no 100% guarantee. Currently, rapidly 
developing genome editing as a method causing

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specific and only minor changes in the genome 
eliminates most potential risks. Nevertheless,

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all GMOs are subject to detailed verification 
of potential risks, so safety should be ensured.

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And now a few words about animal cloning. Cloning, 
in this sense, is a reproductive technique for

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creating genetically identical offspring. However, 
it is often also used for techniques related to

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genetic modification. Cloning techniques 
in mammals include microsurgical embryo

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dissection, isolation and proliferation, 
or aggregation of individual blastomeres,

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and especially nucleus transfer, which may 
or may not be modified. Cloning in mammals

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can be divided into reproductive, where a new 
genetically identical individual is created,

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and therapeutic for the purpose of treatment. The 
first type applies exclusively to animals, while

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the second has great potential in human medicine.
The first successful cloning of a mammal from

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an adult cell was carried out by Professor 
Wilmut of the Roslin Institute in Scotland,

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when the sheep Dolly was born in 1996. The nuclear 
transfer technique was used by cell fusion. Dolly

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was genetically identical to the sheep from which 
the nucleus was taken from a somatic cell of the

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mammary gland epithelium. Interestingly, 
Dolly had three mothers: the first donated

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the genetic information, the second provided an 
empty egg, and the third carried the offspring.

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A number of problems have arisen in connection 
with animal cloning. Low pregnancy rates,

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developmental defects such as early miscarriage, 
stillbirths, early deaths after birth, short

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lifespan, obesity, malformations of various 
organs, and poor immunity have been observed

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in many animals. Cloned animals are generally 
not accepted by breeders - for example, horses

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are not included in studbooks. Legislative and 
ethical problems arise. Regarding food products

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from cloned animals (also known as "cloned 
meat"), the U.S. Food and Drug Administration

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(FDA) states that consuming meat from cloned 
animals is without risk. However, economically,

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this breeding is currently highly inefficient due 
to the high costs of cloning. The European Food

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Safety Authority (EFSA) also declared products of 
animal origin from clones to be safe, but there

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are concerns about the welfare of surrogate 
mothers and the cloned animals themselves.

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A large number of different animal species 
have been cloned, from model organisms such

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as mice and rats to most livestock. The picture 
shows 6 clones of one mare, which the owner rode

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during horse polo. Interestingly, although 
the mares are highly similar, they are not

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genetically identical in terms of coloring. The 
distribution and size of color markings in this

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case are not directly genetically determined. 
It can be said that cloning is often done for

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money or for entertainment or publicity purposes.
I see the main significance of cloning techniques

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primarily in the potential for so-called 
therapeutic cloning, which could enable the

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treatment of previously incurable diseases. 
The mechanism of this treatment involves

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replacing damaged cells with genetically 
identical cells from the patient's own body,

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or cells with corrected genetic information (in 
the case of treatment of genetic diseases). The

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use of embryonic stem cells and, especially, 
induced pluripotent stem cells (iPSCs) created

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directly from the somatic cells of a 
specific patient, appears promising.

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Unlike GMO foods, food from cloned animals 
is more acceptable as it does not contain

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anything foreign. However, both biotechnological 
approaches require improvement in procedures,

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especially with regard to the welfare of the 
lives of these created animals. Neither the

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consumer nor a perfect laboratory analyzer can 
discern the differences between these pieces.

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The issue of GMOs and animal cloning is 
controversial, and everyone must form

0:23:22.880,0:23:28.680
their own opinion on these techniques. 
The aim of this short presentation was

0:23:28.680,0:23:37.120
to provide objective information to help form 
that opinion. Thank you all for your attention.