Transgenic Mice and other Animals. One of the first reports of transgenic animals published in December, 1982, involved the transfer of the growth hormone (GH) gene (from rat) fused to the promoter for the mouse metallothionein 1 (MT) gene. Since then many transgenic animals, including those in cattle, sheep, goats, pigs, rabbits, chickens and fish have been produced and will be utilized in future for a variety of purposes including
(i) their efficiency in utilizing feed;
(ii) ability to give leaner meat,
(iii) their ability to grow to marketable size sooner and
(iv) their resistance to certain diseases.
More than this recently efforts are being made to use transgenic animals as living bioreactors. Transgenic animals produced for this purpose will secrete valuable recombinant proteins and pharmaceuticals into their milk, blood and urine which can be used for extraction of these drugs. This new possibility of manufacturing drugs through transgenic animals is often described as ‘molecular farming’ or ‘molecular pharming’.
Gene abbreviations – ALV = avian leukosis virus; alAT = Al anti trypsin; BPV = bovine papilloma virus; Eu = immunoglobulin heavy chain; FIX = factor IX: GH = growth hormone; GRF= growth releasing factor, β Gal = β galactosidase; hygo = hygromycin; BLG = β – lactoglobulin; MT= metallothionein; MLV= moloney murine leukemia virus; c-myc = a cellular proto oncogene; REV = reticuloendotheliosis; PRL = prolactin; SV= SV = SV 40 TK = thymidine kinase; AFP = antifreeze protein; tPA = human tissue-type plasminogen activator.
Although initially many experiments leading to the production of transgenic animals did not give commercially attractive results, success has been obtained in some recent cases. Some of these cases will be discussed here.
Transgenic goats were successfully produced recently by groups headed by John McPherson and Karl Ebert, both from the USA. These transgenic goats expressed a heterologous protein (a variant of human tissue-type plasminogen activator = LAtPA) in their milk. This protein is used for dissolving blood clots ie., for the treatment of coronary thrombosis. A cDNA representing LAtPA was linked with either the murine whey acid promoter (WAP) or a β casein promoter in an expression vector and used for injecting early embryos obtained surgically from the oviducts of superovulated dairy goats. These injected embryos were either immediately transferred to the oviducts of recipient females (surrogate mothers) or cultured for 72 hours (blocked at 8-16 cell stage), before transfer to the uterus or recipient females of 29 offsprings from 36 recipients, one male and one female contained the transgene. The transgenic female delivered five offsprings, one of which was transgenic showing expression of LAtPA at a low level of few milligrams per litre of milk. In another case of a transgenic goat, few grams of LAtPA per litre of milk could be obtained. At this concentration, the dairy goat may become an economically viable bioreactor for human pharmaceuticals.
In most earlier attempts (in Canada) for the production of transgenic cows, embryos or fertilized oocytes produced in vivo were utilized. Fertilized oocytes or proembryos were surgically terrieved from superovulated and artificially inseminated cows. Microinjected zygotes were than transferred by surgery either directly into the oviduct of recipient cows or into temporary hosts like sheep or rabbits. In view of two surgical operations, this method is labour intensive and more expensive.
In ‘the Netherland’, recently (1991) a technique has been developed for in vitro embryo production. In this new procedure, oocytes obtained from the ovaries of slaughter house cows, were matured and fertilized in vitro. Their pronuclei were microinjected with a construct containing a bovine alpha – Sl casei promoter (bovine = ox) driving a cDNA encoding the antibacterial human iron-binding protein, ‘lactoferrin’. The embryos were cultured to the morula/ blastula stage and then non-surgically transferred to recipient females. Two of the 19 calves born from 103 transferred zygotes were transgenic (one male and other female). This procedure may facilitate the use of cows as bioreactors at the commercial level.
Alilempis to produce transgenic fish started in 1985 and some encouraging results have been obtained. The genes that have been introduced by microinjection in fish include the following:
(i) human or rat gene for growth hormone,
(ii) chicken gene for delta-crystalline protein,
(iii) E. coli gene for β-galactosidase,
(iv) E.coli gene for neomycin resistance,
(v) winter flounder gene for antifreeze protein (flounder-flat fish),
(vi) rainbow trout gene for growth hormone.
The technique of microinjection has been successfully used to generate transgenic in many species such as common carp, catfish, goldfish, loach, medaka, salmon, Tilapia, rainbow trout and zebrafish. In other animals (e.g., mice, cows, pigs, sheep and rabbits), usually direct microinjection of cloned DNA into male pronuclei of fertilized eggs has proved very successful, but in most fish species studied so far, pronuclei cannot be easily visualized (except in medaka), so that the DNA needs to be injected into the cytoplasm.
Eggs and sperms from mature individuals are collected and placed into a separate dry container. Fertilization is initiated by adding water and sperm to eggs, with gentle stirring to facilitate the fertilization process. Egg shells are hardened in water. About 10° to 10° molecules of linearized DNA in a volume of 20 ml or less are microinjected into each egg (1-4 cells stage) within the first few hours after fertilization. Following microinjection, eggs are incubated in appropriate hatching trays, and dead embryos are removed daily.
Since in fish, fertilization is external, in vitro culturing of embryos and their subsequent transfer into foster mothers (required in mammalian systems) is not required. Further, the injection into the cytoplasm is not as harmful as that into the nucleus, so that the survival rate in fish is much higher (35% to 80%).
Human growth hormone gene transferred to transgenic fish allowed growth that was twice the size for their corresponding non-transgenic fish (goldfish, rainbow trout, salmon). Similarly, antifreeze protein (AFP) gene was transferred in several cases and its expression was studied in transgenic salmon. It was shown that the level of AFP gene expression is still too low to provide protection against freeze. There is also a report (in 1991) of the production of transgenic zebrafish from an Indian Laboratory (Madurai Kamraj University). In this attempt, a plasmid containing rat growth hormone gene was microinjected into fertilized zebrafish eggs, and its presence confirmed in adult fish.
The efficiency of the production of transgenic pigs is still very low compared to that of the production of transgenic mice. In mice, 2.5% to 6% of the microinjected eggs developed into transgenic mice, but in pigs this frequency was as low as 0.6% even when as many as 7000 eggs were injected. Despite this low frequency, transgenic pigs carrying growth hormone (GH) gene from bovine (of ‘ox’ origin) of human, and sheep globin gene have been produced (by V.G. Purse) at Agriculture Research Service, Beltsville, USA. The pigs carrying hGH gene showed different levels of expression and only 66% of these animals showed detectable levels of hGH and bGH in their plasma. The animals grew a little faster but did not become large; similarly pigs with sheep globin gene did not show any expression of the transgene for unknown reasons. In these transgenic pigs, however, a modest increase of 10-15% in daily weight and 16-18% in feed efficiency was observed, which is though lower to those in mice, but are comparable to those obtained due to daily injection with pig growth hormone. It was also observed that there was a marked reduction in the subcutaneous fat in some of these transgenic pigs suggesting the possibility of producing leaner meat with lower fat content. These results may have a significant impact on the 9.5 billion dollar annual pig industry in USA.
It is also reported that a long term elevation of growth hormone was generally detrimental to health. The pigs had incidence of gastric ulcers, arthritis and several other diseases. Therefore, techniques will have to be developed to manipulate better the transgene expression by a variety of methods (e.g., changing genetic background or modifying the husbandry regiment).
The rate of transgenesis in sheep is very low (0.1 to 0.2%). This can be improved, if only transgenic viable embryos (after necessary checking) are transferred to surrogate ewes (female sheep). Embryos at 8-16 cell stage can be split into two parts, one for continued culture and the other for detection of integrated genes using polymerase chain reaction (PCR). Although microinjection is the most common method for DNA delivery, gene targeting may be increasingly used in the future. In this approach, embryonic stem (ES) cells in culture are transferred with a vector that targets the gene to a particular site by homologous recombination (as discussed above). The technique, though successfully used in mice, has yet to be applied to sheep, where ES cells will have to be isolated first.
The first reports of transgenic sheep were published by J.P. Simons (1988) of Edinburgh. Two transgenic ewes were produced, each carrying about 10 copies of human anti-hemophilic factor IX gene (cDNA) fused with the 10.5 kb BLG gene (BLG = β -lactoglobulin). BLG gene was used, because it is necessary for specific expression of gene in mammary glands. Consequently, the gene had a tissue specific expression and ewes secreted human factor IX (or alpha-1 antitrypsin) into their milk; this human factor IX is active, even though the expression of transgenic is low. The transgenic ewes were born in early summer of 1986 and were successfully mated same year in December. In 1987, each ewes gave birth to a single lamb. Each lamb inherited BLG-F IX transgene and secreted factor IX in the milk. This programme of the production of transgenic animals by J.P. Simons at Edinburgh was funded by Pharmaceutical Proteins Ltd. (Cambridge, UK) due to its commercial appeal.
In another report (published in 1991), also from Edinburgh, five transgenic sheep were produced (Alan Colman and colleagues). In all these cases, transgene involved fusion of the ovine β-lactoglobulin gene promoter fused to the human aq, antitrypsin (ha1 AT) gene. Four of these animals were female and one male. In one female the protein hATha1 AT reached a level of 35 grams per litre of milk. The protein purified from milk had a biological activity indistinguishable from human plasma derived antitrypsin. The deficiency for ha1AT leads to a lethal disease emphysema, (BC-22), which is a common hereditary disorder among caucasian males of European descent. Therefore any strategy giving high yield of this protein economically will be most welcome. In view of this, transgenic sheep with ha1AT gene will prove very useful as a bioreactor.
Recombinant DNA technique can also be used to increase the ability of sheep for wool growth. For this purpose, genes essential for synthesis of some important amino acids found in keratin proteins of wool, have been cloned and introduced in embryos to produce transgenic sheep. For instance, genes (cysE and cysM) for two enzymes (serine acetyl transferase = SAT and O-acetylserine sulphydryase = OAS), involved in cystein biosynthesis, were isolated from bacteria and cloned in a vector. These genes were introduced in sheep cells, ultimately leading to the production of transgenic sheep, where these genes are expressed. Growth hormone (GH) genes have also been introduced and can be used to promote body weight. Other genes involved in wool production have also been cloned and well be used for transgenesis, thus increasing the potential of wool production through genetic engineering.
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