Transgenic Animals

I. What is transgenic animal?

I.1. Definition

A transgenic animal is one that carry foreign gene that has been deliberately inherited into its genome (the entire set of chromosome) by means of a recombinant DNA technology. In order for all the cells to have the same modification in genetic materials, the transmission must occur through a germ line cell.

This combination includes cutting and splicing of different DNA fragments in order to generate a recombinant DNA to be transferred to a host cell where it can be expressed there.

I.2. History of transgenesis

Before the development of molecular genetics, scientist were used to study the function of gene and its regulation through observation of inherited characteristics and spontaneous mutation, and inorder to enhance the expression of certain traits, they used to selectively breed organisms with each other .

Genetic engineering (as a direct transfer of DNA from one organism to another) first accomplished in 1970 by the production of chimeric mouse (a mouse that contains mixture of genetically different cells). From that time, transgenic techniques start to be applied with the DNA–micro injection followed by the retrovirus mediated transgene and embryonic stem cell mediated gene transfer. These were first applied to mouse and later to a various species such as rats, sheep, gouts, pigs, birds, chicken, fishes, and rabbits.

By the 1980, the first transgene mouse was created by Grandon and Raddle through a pronuclear injection of fertilized egg, and then the use of transgenic animal increases as its widely used for technology in a agriculture, in medical industries, therapeutics and pharmaceutical use.

 

I.3. Transgenic methodology:

As mentioned above, different ways could be applied to generate transgenic animal (transgenic mouse).

a)      injection into the oocyte pronucleus

It’s the first and one of the most used method in transgenic animal creation. In this method, single gene or combination of genes (from same or different species) is directly injected into an oocyte pronucleus of a fertilized egg (Figure 1)

This inserted DNA can increase or decrease the expression of certain genes, or it may be inserted in non-coding regions of a chromosome and therefore not expressed.

Since the process is random, the inserted gene has high probability to be inserted in the target host DNA permitting its expression. This method is applicable to a wide variety of species, which makes it the most commonly used method in transgenesis

b)     Micro  injection into ES cells

Because the isolation of embryonic cell is more difficult than pronucleus of fertilized egg, this method is a complex method (to prepare this EC cells, first we should fertilize the egg in vitro and then culture in the fibroblast). To work on theses EC cells they should pass for one microinjection, and they should be in the log phase. Once these cells prepared and the DNA needed for injection is also ready, we are then able to microinject this later in the EC cells and culture to blastocyte stage. Finally we can implant it into a surrogate mother.

c)    DNA homologous recombination with ES cells

One more method to form transgenic animal is the DNA homologous recombination with ES cells. In this method, plasmid DNA is injected into an in vitro culture of ES, this DNA exchanges place with a DNA present on a sister chromatid (a process of crossing over that we often see during meiosis). First, DNA is incorporated in the EC cells, and these later are then injected into an emberyo during the blastocyte stage. This results in a chimeric animal (see figure 2).

This method is a method of gene interaction were the transferred gene knocked out the process of another gene. Because this technique is capable of knocking out the expression of specific proteins, it's then useful in studying the genetic control.

Figure 2: Diagram displays Methods 1 and 2 of the Embryonic Stem Cell Method for Making a Transgenic Animal

d) DNA can be delivered to the host either chemically or by virus 

1. DNA viral delivery into ES cells

This is also called a retrovirus mediated gene transfer, because the gene to be transferred is through a virus or a plasmid. Retroviruses are RNA viruses that carry reverse transcriptase enzyme to copy RNA into DNA. Progeny derived from this method are chimeric. If the DNA integrate the germ cells, then all of the progeny are transgenic (these could be preserved by freezing for subsequent implantation).

2. DNA chemical Delivery into ES cells 

This method is very similar to the DNA viral delivery. However, the carrier (vector may be) that is responsible to carry the genetic information is a chemical opposed to a retrovirus.

II. The transgenic animal's aplications

The idea of creating transgenic animals gives researchers the opportunity to know the function of certain gene product. One example is the transgenic animal created to study hepatitis B infection. These created animals have the infection antigen, and as the antigen expression increase, several harmful effects lead to severe liver damage and inflammatory response. In addition to using transgenic animal in reganestic study, transgenic animals are useful in agriculture.

II.1. Aggricultural application

By means of transgenesis, a larger cross breading of desired traits can be formed. This can be done by selective cross breeding where farmers used to selectively breed animals to give rise of progeny that exhibit a desired trait (transgenic caw that produces more milk). This can be done in a short time with a high precission. Infact, farmers work on increasing the amount of a specific trait or increasing the quality of these animals ex. the transgenic caw can be made to produce more milk or milk with less lactose/cholesterol L 2. Transgenic plant is another example where they are crossed and efficiently allows farmers to select a desired trait. This can reduce the looseness of crops and improve the growth of the produced plants. However transgenic animals use is not limited to agriculture, it further works in many medical applications.

II.2. Medical application

 Xenotransplantation, nutritional supplements, and gene therapy, are all applications where transgenic animals can be used in.

 II.2.1. Xenotransplantation

Many people die yearly because they fail to find a compatible body organ that suits them. In order to prevent human rejection to any transplant, pigs transgenic for human leukocytes are produced, and corrections in incompatibilities have been generated when organ used in transplantation.

Cancer and human autoimmune disease, are treated by human monoclonal antibodies provided by transgenic mice. These antibodies are cheaper and and can provide a greater yield than the mammalian cells in culture.

II.2.2.  Nutritional suppliments

Many products such as insulin, blood factor, growth hormone, and many other are now provided by milk obtained from a transgenic caws and sheep. This milk is very useful in medicine, where it is used to treat many diseases such as phenylketonuria, cystic fibrosis.

"Rose", is the first transgenic caw used to supply milk used with a specific nutrition or digestive needs, usually given to babies and early people, this milk contains  alpha-lactalbumin.

II.2.3. human gene therapy:

Transgenic animals have a very important role in gene therapy where the normal gene copy is supplied to organism which has this gene mutant or deficient. "Calf" is a transgenic animal produced by AI.Virtaness Institute. This animal carry a gene that encode for a substance responsible for promoting RBCs growth in human. One more example is the sickle cell anemia, a disease cause of point mutation in a beta globin subunit at the gene level (a component of hemoglobin). This can be treated by the supply of the normal copy of the gene .

III. Genetic engineering of animals: Ethical issue including animal welfare concerns

The genetic engineering of animals has been increased significantly in recent years, and the use of this technology brings it with ethical issue some of which related to animal welfare. During the development of Canadian Council of Animal Care (CCAC) guide lines on genetically engineered animals, some of these ethical issues were identified:

1)    Invasiveness of procedures

2)    Large number of animal required

3)    Un anticipated welfare concerns

4)    How establish ethical issue for genetic engineering

Invasiveness of procedures

The generation of genetically engineered animal often involves surgical procedures such as vasectomy, or the scarification of these animals.

During their creation (transgenic animals), oocyte and blastocyte of donor female are induced for super ovulation via hormonal injection, and then the genetically engineered embryos are implanted to a female recipient where the embryo can grow to full term. However, this requires surgery. Males may be vasectomized and used to induce pseudo pregnancy in the female embryo recipient. Then, all of the off springs need to be genotyped which requires taking tissue samples. Hence, progress is being made to turn the methods less invasive and more feasible.  A technique called non-surgical embryo transfer (NSET), somehow like artificial insemination in acting, and can be performed without invasive surgery. Other methods, such as the "Deathless Trans genesis" is more feasible. In this method DNA is introduced to the sperm cell of alive male and there is no need to euthanize female (in order to obtain germ line transmission of genetic alteration). Also the use of PCR in genotyping requires less tissue than southern blot analysis .

Large number of animal required

Because many of the genetically engineered embryos do not survive and of those survive only small proportion carry the correct genetic alteration of our interest, large number of animals required to obtain animals that are of scientific value. In addition, the development of genetic engineering technology has led to large number and variety of transgenic animals. A key refinement to this is the preservation of transgenic animals through freezing embryos or sperm. Further principles of reduction were demonstrated that is once created (transgenic animals) these animals provide a novel and accurate animal models and therefore reduces the total number of transgenic animals used in experiments. But, the variety in available applications and the large number of animal needed for creation, shows that there is still need of ways to decrease the number of animal used in trans genesis.

Unanticipated welfare concern

In fact, genetic engineering has been described as both unpredictable and inefficient process. This later is due to limitation in controlling where DNA should be integrated (the integration site), and thus an increased number of animals that only differs in the integration site is obtained. A refined technique such as gene targeting allows for greater control of DNA integration to avoid unexpected out comes accompanied by these unpredictable interactions with the host. Gene interfering with the host by inserting or removing DNA fragments can result in alteration in the normal genetic homeostasis, for example many of the early produced transgenic animals shows unexpected side effects such as reduced fertility. Further Genetic engineering of the same gene can exhibit different phenotypes and some can produce negative welfare impacts. And because of the difficulty in determining the effect of genetic modification, these animals closely to mitigate any unanticipated welfare concern as it arise.

Once the transgenic animal is created and the welfare concerns are known, the level of monitoring can be reduced if the animal do not exhibits any negative impacts welfare. Hence, with the progress of genetic engineering, new methods could be employed to reduce the unpredictability of the integration site and therefore the procedure may become less welfare.

Beyond animal welfare

Until recently, the main limit to the gene manipulation is technical that is what possible to do, but whether the creation of transgenic animal is acceptable is the question that goes beyond animal welfare consideration. When talking about the nature we should have a look to Arestolilion concept of Telo which describes "essence and purpose of creature". So, one should respect the interest flow from a given telo. Some had made the case that genetic engineering requires us to expand our notion of animal ethics to include concept of the intrinsic value of animals. According to Schicktanz: "human-animal relationship may be damaged by creating a transgenic animal and this is due to the imbalanced distribution (asymmetry) of power between human and animal, which is described as a trouble shift relationships. However, whether limits can or should be placed in genetic engineering differs between people depending on each one view to the concept. For some, moral principles are aside, since they see it as a logical continuation of selective breeding, or because human life is deemed more important than the animal one. So if trans genesis help us to develop a new medicine, morally we have an obligation to do it. Others put moral principles at a risk. Some do not accept the alteration of telo, for them it’s something that shouldn’t be changed. And some others fear from the upset of natural balance of Eco system that may result from genetic engineering .

Such complexity in views concerning genetic engineering makes the putting of ethical limits difficult to achieve. But whatever the case is, achievements need to be made on this issue especially for those animals that are intended to live outside the laboratory.

Other ethical issues

Also genetic engineering is accompanied by concerns over intellectual created animals, and techniques used to produce them. It's important to say here that whatever the method of creation is, there are restrictions on the methods of their disposal.