Fertilization of mammalian eggs is followed by successive cell divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.
Ovine primary fetal fibroblasts were cotransfected with a neomycin resistance marker gene (neo) and a human coagulation factor IX genomic construct designed for expression of the encoded protein in sheep milk. Two cloned transfectants and a population of neomycin (G418)-resistant cells were used as donors for nuclear transfer to enucleated oocytes. Six transgenic lambs were liveborn: Three produced from cloned cells contained factor IX and neo transgenes, whereas three produced from the uncloned population contained the marker gene only. Somatic cells can therefore be subjected to genetic manipulation in vitro and produce viable animals by nuclear transfer. Production of transgenic sheep by nuclear transfer requires fewer than half the animals needed for pronuclear microinjection.
Pancreatic ductal adenocarcinoma (PDAC) is almost universally fatal. The annual number of deaths equals the number of newly diagnosed cases, despite maximal treatment. The overall 5-year survival rate of <5% has remained stubbornly unchanged over the last 30 years, despite tremendous efforts in preclinical and clinical science. There is unquestionably an urgent need to further improve our understanding of pancreatic cancer biology, treatment response and relapse, and to identify novel therapeutic targets. Rigorous research in the field has uncovered genetic aberrations that occur during PDAC development and progression. In most cases, PDAC is initiated by oncogenic mutant KRAS, which has been shown to drive pancreatic neoplasia. However, all attempts to target KRAS directly have failed in the clinic and KRAS is widely assumed to be undruggable. This has led to intense efforts to identify druggable critical downstream targets and nodes orchestrated by mutationally activated KRAS. This includes context-specific KRAS effector pathways, synthetic lethal interaction partners and KRAS-driven metabolic changes. Here, we review recent advances in oncogenic KRAS signalling and discuss how these might benefit PDAC treatment in the future.
Oncogenic Kras activates a plethora of signaling pathways, but our understanding of critical Ras effectors is still very limited. We show that cell-autonomous phosphoinositide 3-kinase (PI3K) and 3-phosphoinositide-dependent protein kinase 1 (PDK1), but not Craf, are key effectors of oncogenic Kras in the pancreas, mediating cell plasticity, acinar-to-ductal metaplasia (ADM), and pancreatic ductal adenocarcinoma (PDAC) formation. This contrasts with Kras-driven non-small cell lung cancer, where signaling via Craf, but not PDK1, is an essential tumor-initiating event. These in vivo genetic studies together with pharmacologic treatment studies in models of human ADM and PDAC demonstrate tissue-specific differences of oncogenic Kras signaling and define PI3K/PDK1 as a suitable target for therapeutic intervention specifically in PDAC.
Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre-loxP–based models. We have developed an inducible dual-recombinase system by combining flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carcinogenesis, genetic manipulation of tumor subpopulations (such as cancer stem cells), selective targeting of the tumor microenvironment and genetic validation of therapeutic targets in autochthonous tumors on a genome-wide scale. As a proof of concept, we performed tumor cell–autonomous and nonautonomous targeting, recapitulated hallmarks of human multistep carcinogenesis, validated genetic therapy by 3-phosphoinositide-dependent protein kinase inactivation as well as cancer cell depletion and show that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are dispensable for tumor formation.
Experimental insertion of a retrovirus into the germ line of mice has resulted in an embryonic recessive lethal mutation. Integration of the proviral genome occurred at the 5' end of the alpha 1(I) collagen gene, leading to complete transcriptional block. Developmental arrest of embryos homozygous at the mutated allele coincides with high expression of the gene in normal embryogenesis. Insertion mutagenesis by retroviruses may offer a general approach to the identification and isolation of genes which are transcriptionally active during mammalian development.
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