Articles by Martin F. Berthelsen in JoVE
Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration Maria Riedel1, Martin F. Berthelsen1, Latifa Bakiri2, Erwin F. Wagner2, Martin K. Thomsen1 1Department of Clinical Medicine, Aarhus University, 2GDD, Cancer Cell Biology Program, National Cancer Research Center (CNIO) This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.
Other articles by Martin F. Berthelsen on PubMed
Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines As an Alternative to the Flp-in System PloS One. | Pubmed ID: 27541869 Recombinase mediated cassette exchange (RMCE) is a powerful tool for targeted insertion of transgenes. Here we describe non-proprietary 'RMCE-in' cell lines as an alternative to the 'Flp-in' system and cell lines. RMCE-in cell lines offer a number of advantages including increased efficiency of integration of the genetic element of interest (GEI) at a single docking site, lack of bacterial backbone at the docking site both before and after GEI integration, removal of selection and visual markers initially present at the docking site upon GEI integration and the possibility to validate GEI integration by loss of a red fluorescence reporter. Moreover, the RMCE-in cell lines are compatible with GEI donors used for the Flp-in system. We demonstrate a three-step procedure for generating RMCE-in cell lines, (I) RMCE-in transposon and SB10 transposase transfection, (II) clone isolation, and (III) selecting single integrated clones with highest RFP level, which could in principle be used to turn any cell line into an RMCE-in cell line. The RMCE-in system was used as a proof of concept to produce three new RMCE-in cell lines using HEK293, HeLa, and murine embryonic stem (mES) cells. The established RMCE-in cell lines and vector are freely available from the ATCC cell bank and Addgene respectively.
Pancreas Specific Expression of Oncogenes in a Porcine Model Transgenic Research. | Pubmed ID: 28664456 Pancreatic cancer is the fourth leading course of cancer death and early detection of the disease is crucial for successful treatment. However, pancreatic cancer is difficult to detect in its earliest stages and once symptoms appear, the cancer has often progressed beyond possibility for curing. Research into the disease has been hampered by the lack of good models. We have generated a porcine model of pancreatic cancer with use of transgenic overexpression of an oncogene cassette containing MYC, KRAS and SV40 LT. The expression was initiated from a modified Pdx-1 promoter during embryogenesis in a subset of pancreatic epithelial cells. Furthermore, cells expressing the oncogenes also expressed a yellow fluorescent protein (mVenus) and an inducible negative regulator protein (rtTR-KRAB). Cells where the Pdx-1 promoter had not been activated, expressed a red fluorescent protein (Katushka). In vitro analyses of cells obtained from the transgenic pigs showed increased proliferation and expression of the transgenes when activated. Induction of the repressor protein eliminated the oncogene expression and decreased cell proliferation. In vivo analysis identified foci of pancreatic cells expressing the oncogenes at day zero post farrowing. These populations expanded and formed hyperplastic foci, with beginning abnormality at day 45. Cells in the foci expressed the oncogenic proteins and the majority of the cells were positive for the proliferation marker, Ki67. We predict that this model could be used for advanced studies in pancreatic cancer in a large animal model with focus on early detection, treatment, and identification of new biomarkers.
A Genetically Inducible Porcine Model of Intestinal Cancer Molecular Oncology. | Pubmed ID: 28881081 Transgenic porcine cancer models bring novel possibilities for research. Their physical similarities with humans enable the use of surgical procedures and treatment approaches used for patients, which facilitates clinical translation. Here, we aimed to develop an inducible oncopig model of intestinal cancer. Transgenic (TG) minipigs were generated using somatic cell nuclear transfer by handmade cloning. The pigs encode two TG cassettes: (a) an Flp recombinase-inducible oncogene cassette containing KRAS-G12D, cMYC, SV40LT - which inhibits p53 - and pRB and (b) a 4-hydroxytamoxifen (4-OHT)-inducible Flp recombinase activator cassette controlled by the intestinal epithelium-specific villin promoter. Thirteen viable transgenic minipigs were born. The ability of 4-OHT to activate the oncogene cassette was confirmed in vitro in TG colonic organoids and ex vivo in tissue biopsies obtained by colonoscopy. In order to provide proof of principle that the oncogene cassette could also successfully be activated in vivo, three pigs were perorally treated with 400 mg tamoxifen for 2 × 5 days. After two months, one pig developed a duodenal neuroendocrine carcinoma with a lymph node metastasis. Molecular analysis of the carcinoma and metastasis confirmed activation of the oncogene cassette. No tumor formation was observed in untreated TG pigs or in the remaining two treated pigs. The latter indicates that tamoxifen delivery can probably be improved. In summary, we have generated a novel inducible oncopig model of intestinal cancer, which has the ability to form metastatic disease already two months after induction. The model may be helpful in bridging the gap between basic research and clinical usage. It opens new venues for longitudinal studies of tumor development and evolution, for preclinical assessment of new anticancer regimens, for pharmacology and toxicology assessments, as well as for studies into biological mechanisms of tumor formation and metastasis.