Production of Transgenic Animals using Virus as a Vector

Read this article to learn about the production of transgenic animals using virus as a vector.

A review of the literature shows that to date the only successful method to produce transgenic rabbits, pigs, sheep, goats and cattle is DNA-microinjection technique into the pro-nucleus.

Rabbits:

Rabbits are used as experimental models in gene transfer experiments. In 1985, the successful production of transgenic rabbits was reported, for the first time, and included the growth hormone construct MT-hGH. The rate of degeneration of rabbit zygotes caused by injection was below 10%. The pre-implantation development capacity of injected zygotes is significantly lower compared with control embryos.

Pigs:

Pig zygotes must be centrifuged to show the pro-nucleus. Fifty percent of the centrifuged non-injected zygotes develop in vivo up to the morula or blastocyst stage. After microinjection, 10- 20% development to various stages of embryonic development occurs. Of the injected zygotes, 5.6% to 11% developed and led to the birth of piglets. The integration rate in pigs is approximately 10%. Growth-hormone constructs used in initial experiments led to an expression rate of 50%.

The production of transgenic F1 offspring is possible. In the authors’ own experiments, inheritance of the trans-gene could be proved in two out of five animals. It is not necessary to centrifuge sheep embryos to make the pro-nucleus visible. According to “Nomarski optics”, 80% of the pro-nuclei can be located if a microscope with interference contrast is available. The capacity for in vivo development of sheep zygotes with injection (26 per cent) and without (10 per cent) is half that of pig embryos after similar treatment.

Seven days after in vivo culture of non-treated and non-in vitro cultured sheep zygotes, Rexroad and Wall in 1987 observed a development rate of 86%. An in vitro culture of five hours’ duration reduced this development rate to 65%, and after the injection of a buffer solution a reduction to 42 per cent was observed. 19% developed to the 32-cell stage after injection of DNA solution.

Sheep and Goats:

Until recently, the trans-genes introduced into sheep inserted randomly in the genome and often worked poorly. However, in July 2000, success at inserting a trans-gene into a specific gene locus was reported. The gene was the human gene for alpha 1-antitrypsin, and two of the animals expressed large quantities of the human protein in their milk.

(1) It was done as sheep fibroblasts (connective tissue cells) growing in tissue culture were treated with a vector that contained these segments of DNA

(2) Regions homologous to the sheep COL1 A1 gene. This gene encodes Type 1 collagen (Its absence in humans causes the inherited disease osteogenesis imperfecta). This locus was chosen because fibroblasts secrete large amounts of collagen and thus one would expect the gene to be easily accessible in the chromatin.

A neomycin-resistance gene to aid in isolating those cells that successfully incorporated the vector. The human gene encoding alphal-antitrypsin. Some people inherit two non- or poorly-functioning genes for this protein. Its resulting low level or absence produces the disease Alphal-Antitrypsin Deficiency (A1AD or Alphal).

The main symptoms are damage to the lungs (and sometimes to the liver).

(1) Promoter sites from the beta-Iactoglobulin gene. These promote hormone-driven gene expression in milk-producing cells.

(2) Binding sites for ribosome’s for efficient translation of the mRNAs.

Successfully-transformed cells were then:

(a) Fused with enucleated sheep eggs and

(b) Implanted in the uterus of a ewe (female sheep),

(c) Several embryos survived until their birth, and two young lambs have now lived over a year, (d) When treated with hormones, these two lambs secreted milk containing large amounts of alphal-antitrypsin (650 µg/ml; 50 times higher than previous results using random insertion of the transgene).

The work on transgenic milk production is expensive requiring large facilities for purifying the protein from sheep’s milk. Purification is important because even when 99.9% pure, human patients can develop antibodies against the tiny amounts of sheep proteins that remain.

GTC Bio-therapeutics, won preliminary approval to market a human protein, anti-thrombin, in Europe. Their protein, the first made in a transgenic animal to receive regulatory approval for human therapy and was secreted in the milk of transgenic goats.

Chickens:

Chickens have several advantages over other farm animals:

(i) Grow faster than sheep and goats and large numbers can be grown in close quarters

(ii) Synthesize several grams of protein in the “white” of their eggs.

Two methods have succeeded in producing chickens carrying and expressing foreign genes:

1. Infecting embryos with a viral vector carrying the human gene for a therapeutic protein and promoter sequences that will respond to the signals for making proteins (e.g., lysozyme) in egg white.

2. Transforming rooster sperm with a human gene and the appropriate promoters and checking for any transgenic offspring.

Preliminary results from both methods indicate that it may be possible for chickens to produce as much as 0.1 g of human protein in each egg that they lay. Not only should this cost less than producing therapeutic proteins in culture vessels, but chickens will probably add the correct sugars to glycosylated proteins something that E. coli cannot do.

Transgenic Fish:

Aquatic animals are being engineered to increase aquaculture production, for medical and industrial research, and for ornamental reasons (Fig. 18.7). Genes inserted to promote disease resistance may allow transgenic fish to absorb higher levels of toxic substances, including heavy metals. In turn, consumers of these fish may be ingesting higher amounts of substances such as mercury and selenium.

Transgenic fish that have genes from species such as peanuts or shellfish that are common causes of allergic reactions in humans may prompt allergic reactions in an unsuspecting consumer. Transgenic species may behave much like invasive species when interacting with the natural environment.

They may compete with native species for resources and pose a threat to the genetic diversity of native populations, especially when genetic modifications such as a rapid growth rate offer advantages over slower-developing native species. Despite industry assurances that transgenic fish would be unable to naturally reproduce or significantly threaten the environment, some scientists are far more doubtful.

The sample bill included in this package addresses these concerns by banning the importation, transportation, possession, spawning, incubation, cultivation, or release of aquatic transgenic animals except under a permit.

Fluorescent Cat:

Recently South Korean scientist produced transgenic white Turkish angora cats to glow red under ultraviolet light these cats contain a fluorescent gene for flu protein and expressed under skin. Subsequently they produced a number of cloned cats from the skin cells of transformed mother cat. They proposed that such cat could be beneficial in diagnosis of genetic diseases and also showed a way to produce endangered animal by cloning.