A versatile modular vector system for rapid combinatorial mammalian genetics

Here, we describe the multiple lentiviral expression (MuLE) system that allows multiple genetic alterations to be introduced simultaneously into mammalian cells. We created a toolbox of MuLE vectors that constitute a flexible, modular system for the rapid engineering of complex polycistronic lentiviruses, allowing combinatorial gene overexpression, gene knockdown, Cre-mediated gene deletion, or CRISPR/Cas9-mediated (where CRISPR indicates clustered regularly interspaced short palindromic repeats) gene mutation, together with expression of fluorescent or enzymatic reporters for cellular assays and animal imaging. Examples of tumor engineering were used to illustrate the speed and versatility of performing combinatorial genetics using the MuLE system. By transducing cultured primary mouse cells with single MuLE lentiviruses, we engineered tumors containing up to 5 different genetic alterations, identified genetic dependencies of molecularly defined tumors, conducted genetic interaction screens, and induced the simultaneous CRISPR/Cas9-mediated knockout of 3 tumor-suppressor genes. Intramuscular injection of MuLE viruses expressing oncogenic H-RasG12V together with combinations of knockdowns of the tumor suppressors cyclin-dependent kinase inhibitor 2A (Cdkn2a), transformation-related protein 53 (Trp53), and phosphatase and tensin homolog (Pten) allowed the generation of 3 murine sarcoma models, demonstrating that genetically defined autochthonous tumors can be rapidly generated and quantitatively monitored via direct injection of polycistronic MuLE lentiviruses into mouse tissues. Together, our results demonstrate that the MuLE system provides genetic power for the systematic investigation of the molecular mechanisms that underlie human diseases

Business as a Regulatory Leader for Risk Governance? The Compact Initiative for Liability and Redress under the Cartagena Protocol on Biosafety

In March 2008, the six world leading agro-biotechnology companies, presented a private, international instrument for liability and redress to cover the environmental damage caused by genetically modified organisms. The proposal was rejected by governments, who instead adopted a binding supplementary liability and redress protocol to the Cartagena Protocol on Biosafety, with no content transfer from the business initiative. Elaborating on this case study, it is explained how powerful business proposals can turn into a policy failure. Business conflicts are identified as one major explanatory factor. The fragmentation of business interests and the lackof business support for the six major firms’ initiative have discredited the role of corporations as regulatory leaders. Business unity is found to be a decisive, necessary condition for the endorsement of corporate proposals by policymakers

β6‐integrin serves as a novel serum tumor marker for colorectal carcinoma

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer‐related deaths worldwide and the need for novel biomarkers and therapeutic strategies to improve diagnosis and surveillance is obvious. This study aims to identify β6‐integrin (ITGB6) as a novel serum tumor marker for diagnosis, prognosis, and surveillance of CRC. ITGB6 serum levels were validated in retro‐ and prospective CRC patient cohorts. ITGB6 serum levels were analyzed by ELISA. Using an initial cohort of 60 CRC patients, we found that ITGB6 is present in the serum of CRC, but not in non‐CRC control patients. A cut‐off of ≥2 ng/mL ITGB6 reveals 100% specificity for the presence of metastatic CRC. In an enlarged study cohort of 269 CRC patients, ITGB6 predicted the onset of metastatic disease and was associated with poor prognosis. Those data were confirmed in an independent, prospective cohort consisting of 40 CRC patients. To investigate whether ITGB6 can also be used for tumor surveillance, serum ITGB6‐levels were assessed in 26 CRC patients, pre‐ and post‐surgery, as well as during follow‐up visits. After complete tumor resection, ITGB6 serum levels declined completely. During follow‐up, a new rise in ITGB6 serum levels indicated tumor recurrence or the onset of new metastasis as confirmed by CT scan. ITGB6 was more accurate for prognosis of advanced CRC and for tumor surveillance as the established marker carcinoembryonic antigen (CEA). Our findings identify ITGB6 as a novel serum marker for diagnosis, prognosis, and surveillance of advanced CRC. This might essentially contribute to an optimized patient care