Viable pigs with a conditionally-activated oncogenic KRAS mutation.

Oncogenic mutations of KRAS play a major role in human carcinogenesis. Here we describe viable gene-targeted pigs carrying a latent KRAS (G12D) mutant allele that can be activated by Cre recombination. These have been produced as part of a program to model human cancers in pigs by replicating genetic lesions known to initiate and drive human disease. Cre-activated KRAS (G12D) animals add to a growing set of gene-targeted pigs that includes a Cre-activated oncogenic mutant TP53, a Cre-responsive dual fluorescent reporter and two truncating mutations of APC (adenomatous polyposis coli). These alleles can be combined and activated in various tissues to produce new models for cancer research.

Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multi-factorial analysis of a large data set.

BACKGROUND: Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency. RESULTS: 109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring. CONCLUSION: Although the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.

Distinct functionality of tumor cell-derived gelatinases during formation of liver metastases.

The specific spatiotemporal role of the matrix metalloproteinase 2 (MMP-2) and MMP-9 (gelatinase) during metastasis is still under debate. Host cells have been described as major contributors to these MMPs during metastasis. Here, we show strong overexpression of MMP-2 and MMP-9 by tumor cells of clinical liver specimen of recurrent metachronous metastases, leading us to address the importance of tumor cell-derived MMP-2 or MMP-9 during liver metastasis. Thus far, distinction of their roles was impossible due to lack of inhibitors which can act exclusively on tumor cells or distinguish MMP-2 from MMP-9. We therefore used short hairpin RNA interference technology in the well-established syngeneic L-CI.5s lymphoma model, in which we could analyze the time course of experimental liver colonization (arrest/invasion of single tumor cells, outgrowth, and invasion within the parenchyma) in immunocompetent mice and correlate these steps with MMP-2 or MMP-9 expression levels. In parental tumor cells, MMP-9 expression closely correlated with the invasive phases of liver colonization, whereas MMP-2 expression remained unaltered. Specific knockdown of MMP-9 revealed a close correlation between invasion-dependent events and tumor cell-derived MMP-9 expression. In contrast, knockdown of MMP-2 did not significantly alter the metastatic potential of the cells but led to a marked inhibition of metastatic foci growth. These findings explain the efficacy of gelatinase-specific synthetic inhibitors on invasion and growth of tumor cells and attribute distinct functions of MMP-2 and MMP-9 to aspects of liver metastasis.