Transgenic animal technology is one of the main and most important parts in biotechnology field. It is used to incorporate exogenous genes into the animal by using different genetic engineering tools that the gene can be expressed and inherited to the offspring. However efficient and accurate control of gene expression is the crucial limiting factors in the development of transgenic animal. There are remarkable events in the history that can be footsteps for today’s knowledge of animal transgenes. The foundation of DNA isolation and gene cloning technology, which help to the study of foreign genes insertion in to mammalian cells continued to utilize for the identification of transfected cells that are successfully combine a new DNA (Grahams and Van der Eb 1973). The development of micromanipulation equipment allows the direct injection of DNA into mammalian cells and these was more efficient than gene transfection method (Diacumakos, 1973), (Capicchi, 1980), (Graessimannet al, 1979). , Jaenisch and Mintz were study on healthy mice by microinjecting SV40 DNA into the blastocysts which were copies of foreign viral DNA (Jaenisch and Mint, 1974) afterward; it was revealed that germline transmission of viral DNA into the animal genome could be attained by directly infecting preimplantation stage embryos with Moloney leukemia viruses (MLV) (Jaenisch, 1976). Such groundbreaking work led to successes of production of transgenic animals.
In 1980 Gordon and his colleagues introduce forieng DNA into mouse embryos by using proneclear microinjection and used the term transgenic for the first time (Gordone et al 1980). Nevertheless his work was not accepted until Palmiter’s demonstration that transgene expression is result phenotypically altered (Palmiter, 1982, 1983). After these consequences a number of research on the potentials of direct DNA injection to develop transgenic animal were conducted. There were trial on three livestock such as Rabbit, Sheep and Pig. However, without centrifugation of livestock embryo visualization of pronucli was difficult and took some time in some animals (Hammer etal1985), (Nanccorrow et al 1987) and (Simons et al, 1988). The other important phenomena were the famous cloned sheep Doly (Wilmut et al., 1997), The first chimeric mice were produced during the 1970s (Brinster, 1990), embryonic stem cell (ES) injection into blastocysts to produce chimera animal (Bradley et al., 1984), sperm-mediated gene transfer (bracket et al.,1971; Lavitrano et al., 1989; Maione et al., 1998), retroviral infection (Chan et al., 1998; Jaenisch, 1976) and also, cloning (Cibelli et al., 1998; Mansour, 1990; Schnieke et al., 1997). These all are the novel transgenic technics and the most important events in these discipline as a whole.
Applications of transgenic
Transgenic science have great role or application in biomedical (human gene therapy, nutritional and pharmaceutical value, xenotransplantation and disease model), genome study (gene function, polymorphism), in agriculture (increased disease resistance, increased growth rate, improved feed utilization, improved milk production and composition, improved carcass composition) and bio pharming.
In agricultural application of transgene, the changing the composition milk that can increase the production of certain protein and growth factor in the deficient milk can improve the survival of newborn animal and human. The comparison of lactation performance of alfa-lactalbumin transgenic and the non transgenic gilts (Nobel et al., 2002) is best model. The development of transgenic swine with growth hormone (Brem et al., 1985; Hamer et al 1985) improves in meat production.
The research in disease and disorder due to genetical mutation are supported by this technology. The transgenic disease model animal created to mimic human disease that can involve the study of symptom, cause, pathways, treatment, and promising way of curing. The most well-known diseases are Alzheimer Mouse (Games et al., 1995), Oncomouse (Thomapson, 2002), Parkinson and other diseases.
The productions of Pharmaceutical protein or antibiotics still another benefit of transgenic technology. This is done through their milk in order to make easier purification, increase production and proteins(Adam, 2003)as a typical example genetically altered goat that produce human antithrombinIII (hAt)which is a serum glycoprotein (Baguisi et al., 1999) preventing from blood clot.
This are some of the application of transgenic technology but it has a huge challenge to apply in concerning the ethical and legality issues from different side religion, animal welfare, food safety, environmental and so on.
Common Technologies of transgenesis
There are various techniques to produce transgenic animal each has its own positive and negative side it need further more study to resolve technical and safety matters. Such approach for transgenic animal production are pronuclear microinjection (Gordone et al., 1980), embryonic stem cell (ES) injection into blastocysts to produce chimera animal (Bradley et al., 1984) sperm-mediated gene transfer(bracket et al.,1971; LAvitrano et al., 1989; Maione et al., 1998), retroviral vector method, (Chan et al., 1998; Jaenisch, 1976) and somatic cell nuclear transplantation method (Cibelli et al., 1998; Mansour, 1990; Schnieke et al., 1997). Others method to improve efficiency integration , gene targeting to improve accuracy, RNA interference-mediated gene silencing technology, zinc-finger nucleases gene targeting technique and induced pluripotent stem cell technology are the parts of the techniques.
DNA pronuclear microinjection has become the most commonly applied method for gene transfer in animals. In 1980 this tool was used to developed transgenic mice for the first time that the technique can help to introduce new genetic material to the recipient animal germ line (Gordon et al., 1980). Technically pronuclear microinjection is injecting a small amount of fluid that contain gene of interest into a male pronuclear of zygote and transfer the zygote to the recipient mother. This procedure seems very simple conversely; it requires skillfulness and generously patient to work appropriately.
The pronucei of many species are clearly visible throughout the lateral phase of the zygotic development. Other species need to centrifuge embryos to displace the optically opaque cytoplasm in order to see the pronuclie of bovine and Swine are example of this species (Wall etal1985). The parameters that used in pronuclear microinjection affect the successful transgenesis such as visualization of pronuclear. Size of DNA, number of transgene copies, DNA isolation and concentration, buffer and time of injection. For instance the isolation and concentration of DNA, they have thie own effect on the survival and viability of embryo(wall et al., 2000) and Brinstor and his partners found that DNA concentration with 10ng/µl highly harmful than DNA concentration 1ng/µl, relatively this 1ng/µl is more effective in integration than 0. 1ng/µ l (Brinstor et al., 1985). AS metioned by (Robert, 2001) the survival rate of embryos injected with DNA purified by a standard agarose gel purification protocol and embryos microinjected with DNA that purified by high speed centrifugation through a NaCl gradient were compared and the standard DNA isolation procedures may result in contaminated DNA which can modify embryo viability.
Microinjection of foreign DNA into pronuclei of fertilized oocytes has been the most successful method for producing transgenic animal. Researchers also tries to improve its efficiency using different means such as restriction enzyme mediated integration (Schiest andPetes,1991), double pronuclear microinjection or injection of both pronuclei (Kupriyanov etal., 1998) and artificial chromosome like BAC,YAC,HAC. However problems including low efficiency, random integration into the host genome, variable expression due to position effects, etc… are the limits it application.
Sperm-mediated gene transfer
Sperm-mediated gene transfer is first demonstrated by (Brackett et al., 1971) that mammalian spermatozoa have the inherent capability to bind exogenous DNA. Lavitrano and co-workers, using mice model, described that such DNA binding ability of sperm could be used to introduce foreign DNA into eggs during fertilization for making transgenic animals (Lavitrano et al, 1989). This report was generated for significant importance of sperm-mediated DNA transfer which was simple and cost effective technique to produce transgenic mice. Though, the difficulties in reproducibility and the lower efficiency in integrating foreign gene into the animal genome resulted in substantial disagreement for several years. But still, several research reports have been published that confirming sperm from many species, including livestock, poultry and fish, can be used as vectors to deliver transgenes into the animal genome (Smith and Spadafora, 2005)
In this technique, DNA was mixed with sperm cells prior to in vitro. The rudimentary approach of sperm-mediated gene transfer is quite forthright. The seminal plasma-free sperm cells are suspended in the proper culture medium, and then incubated with DNA; consequentially DNA-carrying sperms are then castoff to fertilize eggs, through artificial insemination, in the case of aquatic animals like fish, via ordinary fertilization or in vitro fertilization. There are discrepancy in transgenic efficiency has been examined among livestock species in success of sperm-mediated gene transfer. For instance the very efficient DNA transfer was attained in Swine which was up to 80% of the offspring were transgenic (Lavitrano et al., 2002) while in the case of bovine this method had limited achievement to produce transgenic cattle (Schellander et al 1995) therefore, sperm-mediated gene transfer has not been widely adopted for making transgenic animals unlike the other technique.
To improve the efficiency of transgenic, different techniques used that enhances the sperm to capture transgenes. The incorporation of spermmediated gene transfer and intracytoplasmic injection of sperm (ICSI) coated with DNA is the first approach. About 20% of the offspring expressed the integrated exogenous DNA after, membrane-disrupted mouse spermatozoa, incubated with foreign DNA that was microinjected into eggs by ICSI, (Perry, et al., 1999). The limitation of ICSI in some species hinders the application of the technique however; the successful result has been reported only in swine (Lai et al., 2001), (Kurome et al., 2006), (Kato et al., 2004), (Hirabayashi et al., 2005). Electroporation or liposome treatment has been employed to facilitate foreign DNA uptake by sperm (Smith and Spadafora, 2005).
Mouse and pig eggs were fertilized and produced transgenic offspring with high efficiency (Chang et al., 2002 ).this was resulted When a linker-based sperm-mediated gene transfer method used in which DNA was mixed with a monoclonal antibody that recognized a surface antigen on sperm before being incubated with sperm for fertilization. By direct injection of foreign DNA into the reproductive tract of males before mating was also an alternative way for increasing the efficiency of sperm mediated gene transfer (Celebi et al., 2003). As reported by (Shemesh et al., 2000) restriction enzyme mediated insertion method to integrate transgenes into the bull sperm genome was produced transgenic offspring. Generally, all these shows that well established spermatozoa can play a role in transgenesis in all species. Their capability to take up exogenous DNA molecules can be exploited to transmit new genomic information to the offspring after fertilization. This prospective is emphasized by the various developments of SMGT variant protocols.
Embryonic stem cell-mediated gene transfer
ES cells were first established by two laboratories independently in 1981, Embryonic stem (ES) cells are derived from the inner cell mass of mammalian blastocysts with a great capacity to differentiate into cells of all three germ layers and continuing pluripotency (Evans and Kaufman, 1981; Martin, 1981). These three germ layers have the potential to form all of the cell types of the mature animal (muscle, nerve, skin etc.). Embryo stem cells are classified into three type’s embryonic carcinoma (EC) cell, embryonic stem (ES) cell and primordial germ cells or embryonic germ (PGCs) cells. These physiognomies have made them a precious research tool for molecular genetics, disease models and development of biology in mice, human and non-human primates. In the domestic animals, ES cell lines are also useful in the particular genetic engineering of those animals for improved production traits and products, for disease resistance and for bioreactor. Embryonic stem cell lines these animals could be used to achieve either by improving the efficiency of somatic cell nuclear transfer (SCNT) technology that is currently used in the production of genetically modified ungulates (Rideout et al., 2000; Keefer, 2004; Donovan et al., 2005; Wall et al., 2005) or through chimera technology which is well known in producing transgenic mice (Bradley and Robertson, 1986; Bradley, 1987; Wells et al., 2003).
Under certain restricted circumstances mouse ES cells can form trophectodermal cells in vitro (Ralston and Rossant, 2005) and in vivo (Beddington and Robertson, 1989), while marmoset and human ES cells can differentiate without demur into trophectoderm cells (Thomson et al., 1995; Xu et al., 2002). Even though the mouse Embryonic stem cell lines are generated from in vivo-derived embryos are better than in vitro-produced embryos (Bavister, 2004), its efficiencies are low (McWhir et al., 1996). Primate Embryonic stem cell lines have been also established from in vivo-derived blastocysts of monkeys (Thomson et al., 1995) and in vitro fertilized (IVF) in vitro-cultured (IVC) human blastocysts (Thomson et al., 1998; Reubinoff et al., 2000; Lee et al., 2005a). However, strong ES cell line establishment has not yet been proven in either mice or non-human primates. In the case of domestic animals, there is no proven ungulate embryonic stem cell lines currently exist but their embryonic stem-like cell lines were used to as nuclear donor cells generate cloned animals by nuclear transfer (Chen et al., 1999a; Saito et al., 2003). It is impossible to prove embryonic stem cell character of the cells in nuclear cloning character since various types of fully differentiated somatic cell nuclei have confirmed competent for the making of cloned animals (Kato et al., 2000; Wakayama and Yanagimachi, 2001), and, in particular, from trophectoderm cells nuclear cloned animals have been also produced (Tsunoda and Kato, 1998). Additionally trophectoderm cells are a common cell contaminant in attempts to establish ungulate ES cell lines and are easily mystified with epiblast cells.
The ESCs will contribute in the development of host embryo, make chimera till to species chimerism, and transmit the exogenous gene to the subsequent generation and generate transgenic animals (Fig. 2(Li and Bradley, 2011). The level of chimerism can be recognized by considering the coat color of mice after birth. Since the coat color of the parental embryonic cell strain is unrelated to that of the host embryo strain, mice in which both strains contribute to the development will exhibit a mix of two coat colors. This occurrence is characterized as chimerism. The animals showing this trait are called as chimeras. The benefit of carrying out genetic manipulations in embryonic stem cells is that confirmation of the targeted modification can be achieved in vitro before any animal work is begun. One of the chief advantages of embryonic stem celll is that they are comparatively efficient at homologous recombination in contrast to other animal cells. The greatest hindrance for making ES-cell derived transgenic animals is as a result of the technical hitches associated with the production, characterization and maintenance of pluripotent ES cell lines.
On the other hand ES cells are highly efficient material and extensively used in animal cloning. First totipotent stem cells are isolated from the embryo then foreign DNA of interest can be integrated into the cultured ES cells using electroporation which is very efficient method (Xiangyang Miao, 2012) or microinjection. Various markers are used for effective selection of the integrated DNA. The recombinant ES cells are then combined with new blastocyst which can be attained by either blastocyst injection or morula aggregation. Inside the blastocyst they are mixed with the cell of the inner cell mass after that, this blastocyst is implanted into the uterus of foster mother and then pups were produced (Reece, 2004). (Okada et al.2009) used to introduce lentiviral vector to reduce the possibility of random integration and assessed the feasibility of gene targeting in mouse embryonic stem cells mediated by chronic viral vector. The production of genetically modified animals using mouse embryonic stem cell allows Transgenic, knockin, and knockout gene manipulations. For the physiological and pharmacological investigations rats are more important than mice as experimental animals. On the other hand, the production of genetically engineered rats has been difficult because of the problems in rat embryonic stem cells after gene introduction (Kawamata and Ochiya 2011) .This embryonic stem cell mediated technique offers a great opportunity to study the whole cellular function by using modified embryonic stem cells in vitro including the expression extent and stability of the introduced genes. It allows also targeted gene integration which can be challenging in pronucluer microinjection.
Cereals and Cereal Product Microbial Spoilage and Hazards
Cereals, belonging to the Gramineae or the grass family, are the basic carbohydrates sources for the human population (Adams