Transgenic Animals | Zoology Optional Notes for UPSC PDF Download

Introduction

In December 1982, one of the earliest instances of creating transgenic animals involved transferring the growth hormone (GH) gene, derived from rats, fused with the promoter for the mouse metallothionein 1 (MT) gene. Since then, a multitude of transgenic animals, spanning cattle, sheep, goats, pigs, rabbits, chickens, and fish, has been developed.
These animals hold promise for various purposes in the future, such as:

• Enhancing feed efficiency.
• Producing leaner meat.
• Accelerating growth to marketable sizes more quickly.
• Providing resistance to specific diseases.

Furthermore, recent efforts are underway to employ transgenic animals as living bioreactors. Specifically, these animals can be engineered to secrete valuable recombinant proteins and pharmaceuticals into their milk, blood, and urine. These substances can then be extracted for drug production. This innovative approach to manufacturing drugs through transgenic animals is commonly referred to as molecular farming or molecular pharming.

While early experiments in generating transgenic animals didn't always yield commercially attractive results, recent successes have been achieved in some cases, and these instances will be discussed.

Transgenic Sheep

• The transgenesis rate in sheep is currently low (0.1% - 0.2%), but it can be enhanced by transferring only viable transgenic embryos, identified through necessary checks, to surrogate female sheep.
• Embryos at the 8-16 cell stage can be divided into two parts: one for continued culture and the other for detecting integrated genes using polymerase chain reaction (PCR).
• While microinjection is the prevalent method for DNA delivery, there is a potential shift toward using gene targeting in the future. This involves transfecting embryonic stem cells in culture with a vector that targets the gene to a specific site through homologous recombination. This technique, successful in mice, is yet to be applied to sheep, where cells need to be isolated first.
• J.P. Simons of Edinburgh reported the first transgenic sheep. These ewes carried about 10 copies of the human hemophilic factor IX gene fused with the 10.5kb BLG gene, specifically chosen for its role in gene expression in mammary glands. The transgenic ewes, born in 1986, successfully mated in 1986 and each gave birth in 1987 to a single lamb inheriting the BLG-F IX transgene and secreting factor IX in the milk. This project was commercially funded by Pharmaceuticals Proteins Ltd. in the U.K.
• Recombinant DNA techniques can boost sheep wool growth by introducing genes essential for the synthesis of amino acids in keratin proteins. Genes (cysE and cysM) for enzymes involved in cysteine biosynthesis were cloned from bacteria and introduced into sheep cells, resulting in the production of transgenic sheep expressing these genes. Growth hormone (GH) genes have also been introduced to promote body weight, and other genes associated with wool production will be cloned and used for transgenesis to enhance wool production through genetic engineering.

Transgenic Pigs

• The efficiency of transgenic pig production is notably lower compared to transgenic mice. While 2.5% to 6% of microinjected eggs develop into transgenic mice, the frequency in pigs is as low as 0.6%, even with the injection of up to 7,000 eggs. Transgenic pigs carrying a growth hormone gene from bovine or human and a sheep globin gene have been created at the Agriculture Research Service in BeltSalive, USA.
• Pigs with the "hGH" gene displayed varying levels of expression, with only 66% exhibiting detectable levels of human and bovine growth hormones in their plasma. Although these animals grew somewhat faster, they did not reach significantly larger sizes. Similarly, pigs with a sheep globin gene showed no expression of the transgene for unknown reasons.
• Despite the lower frequencies, transgenic pigs exhibited a modest increase of 10 to 15% in daily weight and 16 to 18% in feed efficiency. Although these results are lower than those observed in mice, they are comparable to the effects obtained through daily injection with pig growth hormone.
• Notably, some transgenic pigs showed a marked reduction in subcutaneous fat, suggesting the potential for producing leaner meat with lower fat content.
• The impact of these results on the $9.5 billion annual pig industry in the USA could be significant.
• Long-term elevation of growth hormone was found to be generally detrimental to health in these pigs, leading to issues such as gastric ulcers, arthritis, and other diseases. Consequently, there is a need for the development of techniques to better manipulate transgene expression through various methods.

Transgenic Cows

• In initial attempts to produce transgenic cows, embryos or fertilized oocytes generated in vivo were employed.
• Fertilized oocytes or proembryos were surgically obtained from superovulated and artificially inseminated cows. Microinjected zygotes were then surgically transferred either directly into the oviduct of recipient cows or into temporary hosts like sheep or rabbits. However, due to the need for two surgical procedures, this method is labor-intensive and more expensive.
• A recent technique developed in The Netherlands involves in vitro embryo production. Oocytes obtained from the ovaries of slaughterhouse cows are matured and fertilized in vitro.
• The pronuclei of these embryos are microinjected with a construct containing a bovine alpha S1 casein promoter driving a cDNA encoding the antibacterial human iron-binding protein, lactoferrin.
• The embryos are cultured to the morula/blastula stage and then non-surgically transferred to recipient females. Out of 103 transferred zygotes, two of the 18 calves born were transgenic (one male and one female).
• This procedure holds the potential to facilitate the use of cows as bioreactors on a commercial scale.

Different types of Transgenic fish

• Transgenic fish production efforts commenced in 1985, yielding promising results.
• Genes introduced via microinjection in fish include:
  • Human or rat growth hormone gene
  • Chicken delta crystalline protein gene
  • E. coli beta-galactosidase gene
  • E. coli neomycin resistance gene
  • Winter flounder antifreeze protein gene
  • Rainbow trout growth hormone gene
• Microinjection has successfully generated transgenic fish in species like common carp, catfish, goldfish, and salmon. While direct microinjection of cloned DNA into male pronuclei of fertilized eggs is commonly effective in other animals, many fish species lack easily visualized pronuclei, requiring the collection and placement of DNA into a separate container.
• In fish, fertilization is external, and in vitro culturing of embryos with subsequent transfer into foster mothers is unnecessary. Additionally, injection into the cytoplasm, rather than the nucleus, is less harmful in fish, resulting in higher survival rates.
• Transgenic fish carrying the human growth hormone gene exhibited twice the size of their non-transgenic counterparts.
• Antifreeze protein gene transfer in transgenic salmon revealed that the level of expression was insufficient for freeze protection.
• Reports also mention the production of transgenic zebrafish in an Indian laboratory. A plasmid containing the rat growth hormone gene was microinjected into fertilized zebrafish eggs, and its presence was confirmed in adult fish.
• Ongoing research aims to enhance the qualities of both animals and plants through genetic modification.