R&D pipeline

The mouse, which has long been used as a laboratory animal, is an ideal model on which to study gene function. Nearly twenty years ago the first transgenic mice were produced by inserting transgenes into the mouse genome.

	Brinster RL, Chen HY, Trumbauer ME, Senear AW, Warren R and Palmiter RD Factors affecting the efficiency of introduction foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci (USA), 1985; 82: 4438-4442.
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	DePamphilis ML, Herman SA, Martinez-Salas E, Chalifour LE, Wirak DO, Cupo DY and Miranda M Microinjecting DNA into mouse ova to study DNA replication and gene expression and to produce transgenic animals. Biotechniques, 1988; 6: 662-680.

This technique does not allow the selection of the insertion site or the copy number. Homologous recombination targets the mutation to a locus of interest. This technological breakthrough allows the inactivation of genes (knock-out models) and the targeted insertion of transgenes (knock-in models).

	KR Thomas and MR Capecchi Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell, 1987; 51(3): 503-12.
	Ramirez-Solis, R, Bradley, A Advances in the Use of Embryonic Stem Cell Technology. Curr Opin Biotechnol, 1994; 5: 528-533.
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	Camper SA, Saunders TL, Kendall SK, Keri RA, Seasholtz AF, Gordon DF, Birkmeier TS, Keegan CE, Karolyi IJ, Roller ML. Implementing transgenic and embryonic stem cell technology to study gene expression, cell-cell interactions and gene function. Biol Reprod, 1995; 52: 246-57.
	Müller U Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mechanisms of Development, 1999; 82: 3-21.

Cells containing the modification will transmit the mutation to the whole organism. In certain conditions, such as a deleterious effect of a mutation, it is necessary to limit the modification tissue-specifically. Tissue-restricted mutation has thus been developed (conditional strategies).

	Michaeline Bunting, Kenneth E. Bernstein, Joy M. Greer, Mario R. Capecchi, and Kirk R. Thomas Targeting genes for self-excision in the germ line Genes & Dev., 1999; 13: 1524-1528.
	Zhi-Wei Li, Gerlinde Stark, Jürgen Götz, Thomas Rülicke, Ulrike Müller, and Charles Weissmann Generation of mice with a 200-kb amyloid precursor protein gene deletion by Cre recombinase-mediated site-specific recombination in embryonic stem cells. PNAS, 1996; 93: 6158-6162.
	A Nagy Cre recombinase: the universal reagent for genome tailoring. Genesis, 2000; 26(2): 99-109.
	J Schaft, R Ashery-Padan, F van der Hoeven, P Gruss, and AF Stewart Efficient FLP recombination in mouse ES cells and oocytes. Genesis, 2001; 31(1): 6-10.
	CI Rodríguez, F Buchholz, J Galloway, R Sequerra, J Kasper, R Ayala, AF Stewart, and SM Dymecki High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat Genet, 2000; 25(2): 139-40.

In recent years, some improvements of the previous methods have been developed that allow better gene analysis.

	WC Skarnes, JE Moss, SM Hurtley, and RSP Beddington Capturing Genes Encoding Membrane and Secreted Proteins Important for Mouse Development. PNAS, 1995; 92: 6592-6596
	J Seibler, D Schübeler, S Fiering, M Groudine, and J Bode DNA cassette exchange in ES cells mediated by Flp recombinase: an efficient strategy for repeated modification of tagged loci by marker-free constructs. Biochemistry, 1998; 37(18): 6229-34.
	P Mountford, B Zevnik, A Duwel, J Nichols, M Li, C Dani, M Robertson, I Chambers, and A Smith Dicistronic Targeting Constructs: Reporters and Modifiers of Mammalian Gene Expression. PNAS, 1994; 91: 4303-4307.
	Andreas Kistner, Manfred Gossen, Frank Zimmermann, Jasna Jerecic, Christoph Ullmer, Hermann Lübbert, and Hermann Bujard Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. PNAS, 1996; 93: 10933-10938.
	J Brocard, R Feil, P Chambon, and D Metzger A chimeric Cre recombinase inducible by synthetic,but not by natural ligands of the glucocorticoid receptor. Nucleic Acids Res., 1998; 26(17): 4086-90.

Recently, two techniques have been developed: nuclear transfer and the use of lentiviruses in place of transgene micro-injection.

The nuclear transfer should pave the way for development of gene targeted models in species other than the mouse.
The lentiviruses could provide an efficient system to generate transgenic models in new species, but their use is limited to small-size transgenes

	Teruhiko Wakayama, Ivan Rodriguez, Anthony C. F. Perry, Ryuzo Yanagimachi, and Peter Mombaerts Mice cloned from embryonic stem cells. PNAS, 1999; 96: 14984-14989.
	T Wakayama and R Yanagimachi Cloning of male mice from adult tail-tip cells. Nat Genet, 1999; 22(2): 127-8.
	Alexander Pfeifer, Masahito Ikawa, Yelena Dayn, and Inder M. Verma Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. PNAS, 2002; 99: 2140-2145.

Other references can be found at : http://www.ncbi.nlm.nih.gov/PubMed/