Transgenic animal

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Transgenic animals are animals produced with externally introduced genes. Transgenic animals can be used in many fields and as models to test the effect of certain genes on health. They can be used to produce "enhanced" versions of an animal.

They can also be used as bioreactors: animals that produce an extra substance we want. Imagine having a transgenic cow that is modified to produce insulin in large quantities in its milk. This insulin can then be purified from the cows milk and used in replacement therapy for treating patients with diabetes mellitus.

Introduction

Although several steps in the evolution of transgenic technology had been performed in the 1970´s, transgenesis was not widely recognized before Palmiter et al. [1] introduced the human growth hormone gene into mouse zygotes by pronuclear microinjection and transgenic offspring demonstrated dramatic growth. Since then transgenic techniques have been of immense importance, as they allow new approaches to life science reasearch that general cell culture techniques cannot deliver [2]. The manufacture of large quantities of complex, bioactive proteins like hormones or growth factors for therapeutic purposes (pharming) [3] is only one example of a wide range of different applications for the study of genetic regulation, animal development or disease pathology. For this purpose, foreign DNA is introduced into fertilized oocytes or embryos (blastocysts) of mice, rats and other mammals. [1,4,5]

Production of Transgenic Animals

Transgenic animals are frequently created by two different techniques: microinjection of DNA into the pronucleus of zygotes and injection of embryonic stem cells into blastocysts. The procedure is done with the help of a complete microinjection set-containing of a microscope, micromanipulators, microinjectors and micropipettes.

Microinjection of DNA into the pronucleus

The pronuclear microinjection method of producing a transgenic animal is based on the introduction of linear DNA sequences into the chromosomes of fertilized eggs. The foreign DNA must be integrated into the genome prior to the doubling of the genetic material that precedes the first cleavage in order for the animal to be born with a copy of this new information in every cell. For several hours following the entry of the sperm into the oocyte, the male and the female pronuclei can still be seen individually under a normal light microscope and they have not fused yet into a so called zygote The foreign DNA may be injected into either pronuclei with no difference in results; however, the DNA is typically injected into the male pronucleus because it is slightly larger and closer to the oocyte surface. These oocytes are subsequently transferred into the uterus of pseudopregnant recipient animals. The offspring is screened to confirm a successful integration of the gene of interest for use in further studies.

Injection of ES cells into blastocyst-stage embryos

Embryonic stem cells (ES cells) are derived from the inner cell mass of blastocysts (early embryos). These cells are pluripotent, which means that they can develop into almost any type of tissue. ES cells are used for more precise modifications of the mouse genome. This technique makes it possible to insert as well as remove or modify DNA sequences. Knock-out, knock-in and conditional mutant mice [7] can be produced with this method. The first step is the removal of ES cells from a blastocyst. After transfection of the ES cells, selection, cloning and screening methods make it possible to detect ES cell clones that demonstrate the desired, site-specific recombination. After microinjection of the genetically modified ES cells into blastocyst-stage embryos the ES cells divide and become part of the embryo. The following chimeric animals will subsequently transmit the recombinant genotype to their offspring, if the ES cells have contributed to their germ cells.

Production of tetraploid mouse embryos

tetraploid blastocysts with either embryonic stem (ES) cells or diploid embryos has become an established technique for creating mouse chimeras, e.g., by gene targeting. It thus enables fast and efficient analysis of gene function.In both methods the tetraploid cells are not able to contribute to the embryo itself, but instead create the primitive endoderm derivatives and the trophectoderm [8]. The creation of an ES cell tetraploid embryo chimera, in particular, provides an important research tool. Due to cell complementation, complete segregation of descendants of ES cells and tetraploid cells is achieved, resulting in fetuses and viable offspring that are completely derived from ES cells [9,10,11]. ES cell tetraploid embryo chimeras are employed in several approaches: they enable rapid analysis of mutant phenotypes that are derived from genetically modified ES cells, eliminating time consuming breeding and germ line transmission. Gene functions in the embryonic versus extra-embryonic lineages can be tested, and complex phenotypes can be produced by generating embryos that carry mutations of multiple genes [12]. This method is also the only way to analyze genes known to be lethal to heterozygous embryonic [13].

References

[1] Palmiter R.D., Brinster R.L., Hammer R.E., Trumbauer M.E., Rosenfeld M.G., Birnberg N.C., Evans R.M.: Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. (1982) Nature 300, 611-615

[2] Chan AWS: Transgenic animals: Current and alternative strategies. (1999) Cloning, 1:25-46.

[3] Wall, R.J., Kerr, D.E., and Bondioli, K.R. : Transgenic Dairy Cattle: Genetic Engineering on a Large Scale. (1997) J.Diary Sci. 80,2213-2224

[4] Gordon, J.W., Scangos, G.A., Plotkin, D.J., Barbosa, J.A., Ruddle, F.H.: Genetic transformation of mouse embryo by microinjection of purified DNA. (1980) Proc.Natl.Acad.Sci USA 77, 7380-7384

[5] Hammer RE, Pursel VG, Rexroad CE Jr, Wall RJ, Bolt DJ, Ebert KM, Palmiter RD, Brinster RL: Production of transgenic rabbits, sheep and pigs by microinjection.(1985) Nature 315 (6021), 680-3.

[6] Nagy,A., Gerstenstein, M., Vintersten, K., Behringer, B.: Manipulating the Mouse Embryo, A Laboratory Manual. Third edition (2003) Cold Spring Habor Laboratory Press

[7] Barbinet, C.:Transgenic Mice: An irreplaceable tool for the study of mamalien developmental biology. (2000) J.Am.Soc.Nephrol 11:88-94

[8] Tarkowski, A. K., Witkowska, A. and Opas, J.: Development of cytochalasin Betainduced tetraploid and diploid-tetraploid mosaic mouse embryos. (1977). J. Embryol. Exp. Morphol.41: 47-64.

[9] Nagy, A., Gocza, E, Diaz, E. M., Prideaux V. R., Ivanyi, E. Markkula, M. and Rossant. J.: Embryonic stem cells alone are able to support fetal development in the mouse. (1990). Development 110: 815–821.

[10] Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. and Roder, J.C.: Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. (1993). Proc. Natl. Acad. Sci. USA 90 (18): 9424-9428.

[11] Eggan, K., Akutsu H., Loring J., Jackson-Grusby L., Klemm M.,. Rideout W. M III, Yanagimachi R. and Jaenisch R.: Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. (2001) Proc. Natl. Acad. Sci. USA 98: 6209–6214

[12] Tam, P. P. L. and Rossant, J.: Mouse embryonic chimeras: tools for studying mammalian development. (2003) Development 130: 6155-6163.

[13] Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal, K., Eberhardt, C., Declercq, C., Pawling, J., Moons, L., Collen, D., Risau, W. and Nagy, A.: Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. (1996) Nature 380: 435-439.

[14] Peloquin, J.J., Thibault, S.T., Schouest, L.P., Miller, Jr., Miller, T.A.: Electromechanical Microinjection of Pink Bollworm (Pectinophora gossypiella) Embryos Increases Survival. (1997) BioTechniques 22: 496-499

[15] Al-Hasani, S., Ludwig, M., Karabulut, O., Al-Dimassi, F., Bauer, O., Sturm, R., Kahle, D., Diedrich, K.: Results of intracytoplasmatic sperm injection using the microprocessor controlled TransferMan Eppendorf manipulator system. (1999) MEFS Journal Vol. 4, No.1, 41-44

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