In 1953, after Watson and Crick completed the molecular DNA structure there were many advancements in molecular technology. These developments lead to discoveries involving recombinant DNA, genetic cloning, the analysis of gene expression, and genomic mapping. The knowledge from all these various biological technologies help produce transgenic animals (Margawati, Endang).  

     There are three different methods that would allow for the creation of transgenic animals. The three ways to produce a transgenic animal is through DNA microinjection, retrovirus-mediated gene transfer, and embryonic stem cell- mediated gene transfer (Margawati, Endang). The first successful transgenic animal was created through microinjection and it takes place shortly after the egg has been fertilized. The specified foreign DNA gene is extracted from an organism, by using restriction enzymes.

Restriction enzymes are enzymes that have the ability to recognize a

specific nucleotide sequence and then cut the DNA molecule at the

point (Fraser Douglas, Barry LeDrew, Angela Vavitas, and Meredith

White-McMahon 366). The foreign extracted DNA fragment is injected

into the pronuclei of the fertilized egg. Once the chromosomes begin

to replicate, the foreign DNA is able to merge with the chromosome,

which means that when the oocyte begins to form an embryo, the

extracted gene trait can be found in the daughter cells. The oocyte is

then placed into a pseudopregnant female carrier who then cares the

embryo until birth (DNA Microinjection Services).

                                                                                 The second way to produce a transgenic animal is through a  

                                                                                 process called retrovirus. Retrovirus refers to a virus that  

                                                                                 basically contains genetic information in the form of RNA

                                                                                 instead of DNA. The virus is used to help carry the desired

                                                                                 gene and it uses an enzyme called reverse transcriptase.

                                                                                 This is a viral enzyme that utilizes RNA as a template to

                                                                                 produce a complementary DNA strand. This means that once

                                                                                 the virus is inserted it will create the complementary strand of

                                                                                 RNA. The reverse transcriptase will then create another DNA

                                                                                 strand that is complementary to the first. Both of these DNA

​                                                                                 strands are then integrated into the host cell’s genome  

                                                                                 through the use of an enzyme called integrase (Fraser  

                                                                                 Douglas, Barry LeDrew, Angela Vavitas, and Meredith White-

                                                                                 McMahon 351).

​

 

​

​

     The third method of producing a transgenic animal is
​​through embryonic stem cells. Similar to the other

processes, it is necessary that the desired trait is

retrieved through an organism. Recombinant DNA is

used to allow a virus or plasmid to carry the desired trait

and the recombinant DNA will then be integrated into

the chromosomal structure of the host animal.

Embryonic stem cells are then used as embryonic stem

cells have the ability to specialize into the form of any

cell. They are exposed to the foreign DNA and some of

the embryonic stem cells will carry the foreign DNA.

However, not all the stem cells will incorporate the

foreign DNA, therefore, it is necessary to differentiate

between the cells that do carry the DNA and those

that do not. This is accomplished through the use of an

antibiotic called G418 and an antiviral drug called ganciclovir. The cells that don’t carry the DNA at all, which are most of them, die when they are exposed to G418. There are still some cells that need to be killed with the use of ganciclovir. This leaves very few cells that have been transformed by homologous recombination. Now that the specific cells have been isolated, they are inserted into organism’s blastocyst. Once again, the embryo is transferred into a pseudopregnant female who will carry the embryo until birth (Trasgenic Animals). To check if the transgene is present a method called southern blotting is used. The process of southern blotting starts off with the DNA of the potentially transgenic animal being cut by restriction enzymes and which is then separated by electrophoresis which occurs in agarose gel. This step causes the DNA strands to denature into two single strands. A special blotting paper is used to transfer the DNA fragments onto the membrane and out of the gel, while still maintaining the same separation between fragments. A process known as hybridization is then used, where the DNA fragments are placed in a hybridization bottle containing a radioactive labelled probe solution. The probe binds to the complementary DNA fragments found on the membrane, and the radioactive probe indicates the fragment to which a probe has been attached.