Dr. Syed Abdullah Gilani Animal Biotechnology Dr. Syed Abdullah Gilani

Animal breeding is in practice since long times Animal breeding is in practice since long times. Selective breeding aims to increase the frequency of a large number of genes that work together with the remainder of the animal’s genes or genome to produce the desired phenotypes. Selective breeding increased three times more milk production of the average dairy cow. Selective animal breeding is extremely slow process, especially when dealing larger animals with long gestation periods. It may take many years to establish desired phenotypic changes. The advent and application of recombinant DNA technology in animal breeding programs could greatly increase the speed and range of selective breeding.

Genetic Manipulation and Transgenic Animals

The reasons for producing genetically modified animals To help scientists to identify, isolate and characterise genes in order to understanding more about their function and regulation. To provide research models of human diseases, to develop new drugs and new strategies for repairing digestive genes (‘gene therapy’). To provide organs and tissues for use in human transplant surgery. To produce milk that contains therapeutic proteins; or to alter the composition of the milk to improve its nutritional value for human infants. To enhance livestock improvement programmes

Opportunities that may be envisaged within animal breeding programs: Efficiency of meat production, Improved quality of meat, Milk quality and quantity, Egg production, Wool quality and quantity, Disease resistance in animals, and Production of low-cost pharmaceuticals and biologicals

Pro-nuclear infection Overall strategy for creating transgenic animal is similar to that used to develop a transgenic plant: It is necessary to isolate the desired DNA sequence that codes for the desired protein together with an understanding of promoter, terminator and regulatory regions of the gene. The linearised DNA is then ready for transfer.

At the present time, the most successful method for gene transfer into livestock is by microinjection into the pronucleus of fertilised eggs. Microinjection techniques make use of finely constructed glass needles, which allow the injection of purified DNA into the fertilised eggs of the chosen species: a process known as transfection. The eggs are then surgically transferred into hormonally synchronised surrogate mothers. Unlike mice and pigs, litter size is limited to one or two in sheep and cattle and, therefore, large numbers of animals have to be employed as recipients for the microinjected eggs.

Transgenic pigs, sheep and cattle, and other animals, have now been obtained although the frequency of success is only about 1% compared with 2–5% in mice. This low efficiency of the technology will continue to exert some limitation to wider acceptance. However, with fish the eggs are fertilised externally and thus eliminate many of the complicated techniques required in mammals to harvest ova, fertilise them and then introduce the embryos into foster mothers. Successful fish transgenics can be as high as 70%.

Steps to establish transgenic animals Identification and construction of foreign gene (genetic engineering). Microinjection of DNA directly into the pronucleus of a single fertilised egg. Implantation of these engineered cells into surrogate mothers. Bringing the developing embryo to term. Proving that the foreign DNA has been stably and heritably incorporated into the DNA of at least some of the newborn offspring. Demonstrating that the gene is regulated well enough to function in its new environment.

There have been successes in: The ultimate goal of animal breeders will be to introduce specific economically important traits into commercial livestock. However, the knowledge of the mechanisms regulating gene expression in most higher animals is limited, and this limits the ability to construct transgenic animals. There have been successes in: improving feed efficiency and increasing lean muscle mass, but most international efforts have been directed to the production of compounds mainly for human medical application, and improvement of an animal’s ability to resist disease.

Somatic cell nuclear transfer (SCNT) In the 1950s scientists removed the nuclei from frogs’ eggs and replaced them with somatic nuclei from embryos and succeeded in raising adult frogs. This was then followed by serial transplantations using the nuclei from the transplanted embryos for more transplantation. All the embryos could be considered to have identical nuclei; this was originally termed nuclear cloning, but is now known as somatic cell nuclear transfer (SCNT). In the 1990s, Dolly the sheep was created in Scotland from a nucleus derived from an adult ewe inserted into an enucleated oocyte.

How the dolly is produced by SCNT process? There are two aspects to the SCNT process. A ewe is induced to superovulate and the eggs collected. By means of a micropipette, the cell nucleus (DNA) is siphoned out leaving an enucleated cell, but with a small residual amount of mitochondrial DNA in the cytoplasm. Meanwhile, tissue has been removed from the udder of an adult ewe, the individual cells then cultured axenically (i.e., raised under sterile conditions) on a plate dish and subsequently induced into a resting phase by limited starvation. A single cell, containing a full set of chromosomes, is picked up by a micropipette and transferred into the space between the empty egg and the cytoplasmic outer lining. An electric current is applied and fusion occurs. The resulting embryo or clone is cultured and then transferred into a surrogate mother and in c. 21 weeks a lamb is born.