Generation of a Fanconi anemia models in the pig by advanced genome engineering
Final Report Abstract
In summary, good progress toward the establishment of a Fanconi anemia model in the pig were made, albeit the final aim of a pig with the mutated FANCA gene was not reached. However, the molecular CRISPR/Cas9 tools for targeting of the FANCA gene were produced and tested in porcine cell cultures and parthenogenetic embryos. In addition, a FANCA rescue vector was generated and validated. The cytoplasmic injection of CRISPR/Cas9 plasmids in murine and porcine embryos was validated and the specific targeting of reporter constructs and the FANCA gene was confirmed. Thus the in ovo engineering is a suitable alternative to the commonly applied animal cloning method. Importantly, a syngeneic cohort of piglets carrying two different reporter genes could be generated by re-targeting of a transposon-tagged locus for a site-specific gene knock-in. This approach expands the arsenal of genome engineering technologies in domestic animals, and will facilitate the development of large animal models for human diseases. Potentially, the syngeneic cohort of pigs will be instrumental for vital tracking of transplanted cells in pre-clinical assessments of novel cell therapies.
Publications
-
2015. Exogenous enzymes upgrade transgenesis and genetic engineering of farm animals. Cellular and Molecular Life Sciences 72, 1907-1029
Bosch P, Forcato DO, Alustiza FE, Alessio AP, Fili AE, Olmos-Nicotra MF, Liaudat AC, Rodriguez N, Talluri TR, Kues WA
-
2016. Cytoplasmic injection of murine zygotes with Sleeping Beauty transposon plasmids and minicircles for the generation of germline transgenic mice. Biotechnology Journal 11, 178-184
Garrels W, Talluri TR, Ziegler M, Most I, Forcato DO, Schmeer M, Schleef M, Ivics Z, Kues WA
-
2016. Expression of active fluorophore proteins in the milk of transgenic pigs bypassing the secretory pathway. Scientific Reports 6, 24464
Mukherjee A, Garrels W, Talluri TR, Tiedemann D, Bösze Z, Ivics Z, Kues WA
-
2016. Identification and re-addressing of a transcriptionally permissive locus in the porcine genome. Transgenic Research 25, 63-70
Garrels W, Mukherjee A, Holler S, Cleve N, Talluri TR, Barg-Kues B, Diederich M, Köhler P, Diederich M, Petersen B, Lucas-Hahn, Niemann H, Izsvac Z, Ivics Z, Kues WA
-
2016. One-step multiplex transgenesis via Sleeping Beauty transposition in cattle. Scientific Reports 6, 21953
Garrels W, Talluri TR, Apfelbaum R, Carratala Y, Bosch P, Pötzsch K, Grueso E, Ivics Z, Kues WA
-
Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system. Theriogenology. 2016 Nov;86(8):1886-1896
Bevacqua RJ, Fernandez-Martín R, Savy V, Canel NG, Gismondi MI, Kues WA, Carlson DF, Fahrenkrug SC, Niemann H, Taboga OA, Ferraris S, Salamone DF
-
DE 10 2015 219 318.7 Verfahren zur Herstellung heterologer Proteine in der Milch transgener Tiere. Offenlegung 06.04.2017
Kues, W.A., Garrels, W., Ivics, Z.
-
Strategie zur Erstellung eines Fanconi-Anämie Modells im Schwein mittels Clustered Regularly Interspaced Short Palindromic Repeats (Crispr). University of Veterinary Science Hannover
Ronja Apfelbaum
