Transgenesis & Gene Transfer

Managing PI: Peter Carmeliet - Supervisor: Luc Schoonjans

The function of a gene in biological processes in vivo and its relevance for the pathogenesis of disease can be readily studied by manipulating the genome of animal models (mouse, zebrafish, tadpoles and others). Over the last three decades, the biomedical scientific community has witnessed a (r)evolution in the technical possibilities that have become available to manipulate the genome of these species with great precision, thereby offering unprecedented opportunities to generate disease animal models, elucidate new insight in biological processes in health and disease and propose novel therapeutic concepts and strategies. The latter can be tested by using additional gene manipulation strategies, amongst which gene transfer technologies. The CCB has played a pioneering role in using these technologies to study vascular biology: for instance, they published the first knockout mouse models that were deficient of any type of proteinase or key angiogenic factor, and provided the first genetic evidence, using a knockin strategy, that VEGF also has neuroprotective activities in vivo.


Transgenesis

blastocystCommonly used methods to generate transgenic mice (for transgenesis of zebrafish , we refer to the Zebrafis Facility) are available at the CCB: micronuclear injection of transgenes in zygotes (plasmids as well as BAC transgenes, engineered via BAC recombineering); as well as a series of technologies based on the use of embryonic stem (ES) cells: homologous recombination of targeting vectors to inactivate genes (knockout using plasmid or BAC targeting vectors), modifiy coding or non-coding sequences of genes (knockin), conditionally inactivate genes in tissue-specific manner (Cre/LoxP or Flp/Flt technology; use of tamoxifen-inducible Cre constructs in combination with or without double-fluorescent Cre reporter mT/mG,mouse), conditionally activate genes (targeting of transgenes, preceeded by a floxed stop cassette, into the Rosa26 locus; or tetracycline-inducible rTAT system), set of Cre-mouse lines, etc. Blastocyst injection or aggregation of targeted ES cell clones are routinely used to generate chimeric mice, that are then testbred for germline transmission. Tetraploid aggregation is available to generate completely ES cell-derived embryos. Over 140 different transgenic mouse strains have been generated over the last 22 years. Recently the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 technology is being introduced. It will allow the generation of null, conditional, precisely mutated, reporter, or tagged alleles in mice with unprecedented simplicity and speed.

Several knock-out mouse strains are available for collaborations. Feel free to contact Peter Carmeliet for more information.


Gene Transfer

The mouse genome can be also manipulated via transferring transgenes to somatic cells, using the following techniques, available at the CCB:Transgenesis

  • Non-viral gene transfer: plasmid electroporation (very efficient in skeletal muscle) is routinely used to transiently overexpress transgenes or silence (knockdown) endogenous genes using short hairpin RNA vectors in vivo.
  • Viral gene transfer: adenoviral gene transfer in skeletal muscle or the heart to induce local transgene expression or systemic injection of recombinant adenoviruses to overexpress transgene products in the circulation (after intravenous injection, the adenovirus will transduce predominantly the hepatocytes, which will then function as a production site of the transgene protein); adeno-associated gene transfer using AAV2 and AAV8 is routinely used to overexpress transgenes or silence endogenous genes in skeletal muscle; retro- and lentiviral gene transfers methods are also available (as used for transduction of neuronal cells).

References:

  • The role of fatty acid fl-oxidation in lymphangiogenesis. Wong BW, Wang X, Zecchin A, Thienpont B, Cornelissen I, Kalucka J, et a., Carmeliet P. Nature. 2017 Feb 2;542(7639):49-54.
  • Tumour hypoxia causes DNA hypermethylation by reducing TET activity. Thienpont B, Steinbacher J, Zhao H, D'Anna F, Kuchnio A, et al., Carmeliet P, Lambrechts D. Nature. 2016 Sep 1;537(7618):63-68.
  • Fatty acid carbon is essential for dNTP synthesis in endothelial cells. Schoors S, Bruning U, Missiaen R, Queiroz KC, Borgers G, Elia I, Zecchin A, Cantelmo AR, Christen S, Goveia J, Heggermont W, GoddÈ L, Vinckier S, Van Veldhoven PP, Eelen G, Schoonjans L, Gerhardt H, Dewerchin M, Baes M, De Bock K, Ghesquière B, Lunt SY, Fendt SM, Carmeliet P. Nature. 2015 Apr 9;520(7546):192-7.
  • Role of PFKFB3-driven glycolysis in vessel sprouting. De Bock K, Georgiadou M, Schoors S, et al., Carmeliet P. Cell. 2013 Aug 1;154(3):651-63.
  • VEGF-D deficiency in mice does not affect embryonic or postnatal lymphangiogenesis but reduces lymphatic metastasis. Koch M, Dettori D, Van Nuffelen A, Souffreau J, Marconcini L, Wallays G, Moons L, Bruyère F, Oliviero S, Noel A, Foidart JM, Carmeliet P, Dewerchin M. J Pathol. 2009 Nov;219(3):356-64.
  • Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Oosthuyse B, Moons L, Storkebaum E, Beck H, Nuyens D, Brusselmans K, Van Dorpe J, Hellings P, Gorselink M, Heymans S, Theilmeier G, Dewerchin M, Laudenbach V, Vermylen P, Raat H, Acker T, Vleminckx V, Van Den Bosch L, Cashman N, et al. &, Carmeliet P. Nat Genet. 2001 Jun;28
  • Role of Gas6 in erythropoiesis and anemia in mice. Angelillo-Scherrer A, Burnier L, Lambrechts D, Fish RJ, Tjwa M, Plaisance S, Sugamele R, DeMol M, Martinez-Soria E, Maxwell PH, Lemke G, Goff SP, Matsushima GK, Earp HS, Chanson M, Collen D, Izui S, Schapira M, Conway EM, Carmeliet P. J Clin Invest. 2008 Feb;118(2):583-96