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.

Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.

Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.

Truncation of MIMT1 gene in the PEG3 domain leads to major changes in placental gene expression and stillbirth in cattle.

We previously identified a microdeletion (Del) in the maternally imprinted PEG3 domain in cattle that results in loss of paternal MIMT1 expression and causes late-term abortion and stillbirth. The mutation, when inherited from the sire, is semilethal for his progeny, with 85% mortality. Here we precisely delineate the deletion and describe comparative analyses of fetuses carrying the deletion with wild-type (WT) siblings. Global DNA methylation analysis of cotyledon tissue revealed greater global CpG methylation in fetuses with the deletion (P = 0.003). Gene expression microarray analyses identified increased NPSR1A, IL1RN, NOS3, IL4R, ZDHHC22, and SMOC2 expression in fetuses carrying the deletion and decreased GRID1, PLG, and IGF1 expression. Involvement of the NPSR1A, IL1RN, NOS3, and IL4R genes suggests that some form of restriction related to blood supply, perhaps hypoxemia, may play a role in the pathological mechanism. Also, imprinted genes known to play a role in mammalian prenatal development, specifically IGF2, DLK1, MEST, AST1, PEG3, APEG3, and H19, showed differential expression. The most striking difference was abundant abnormal expression of the neuropeptide S receptor 1 (NPSR1) gene in placental cotyledon tissue of 7 of 11 MIMT1(Del/WT) fetuses. The similarity of this proportion to that of the semilethal mortality rate suggests that abnormal NPSR1 expression may be linked to death or survival of MIMT1(Del/WT) fetuses. NPSR1 is expressed as two isoforms (A and B), and isoform A was detected in MIMT1(Del/WT) cotyledons. NPSR1A is normally not expressed in placenta. Its role in the stillbirth phenomenon has yet to be elucidated, but it may provide a useful prognostic indicator.

Inactivation and inducible oncogenic mutation of p53 in gene targeted pigs.

Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs) and live pigs carrying a latent TP53(R167H) mutant allele, orthologous to oncogenic human mutant TP53(R175H) and mouse Trp53(R172H), that can be activated by Cre recombination. MSCs carrying the latent TP53(R167H) mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53(R167H) allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53(R167H) mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.

RNA interference in pigs: comparison of RNAi test systems and expression vectors.

We have examined the use of RNA interference as a means of downregulating gene expression and provide the first comparison of shRNA and artificial miRNA constructs for transgenic livestock. Several in vitro assays were performed to identify the most effective RNAi constructs. shRNA and miRNA constructs achieved significant downregulation of two porcine target genes: the milk whey protein beta-lactoglobulin and the tumour suppressor p53. Results of different assays were, however, sometimes at variance, indicating that no one assay can be relied upon to predict the effectiveness of an RNAi construct. Our findings are that screening of RNAi constructs is most informative if carried out in primary cells that express the target gene and are competent for somatic cell nuclear transfer. Importantly, the use of miRNA constructs makes tissue specific gene knockdown in large animals a realistic possibility.

Isolation of bacteria from mouse caecal samples and description of Bacteroides sartorii sp. nov.

Caecal samples from wild-type and TNF(deltaARE) mice were cultured on selective media containing bile salts, amino acids or casein macro-peptides. Twenty-two strains were isolated and identified by 16S rRNA gene sequencing. Twenty-one strains showed >98% similarity to known bacteria (Blautia spp., Clostridium innocuum, Enterococcus spp., Escherichia coli, Lactobacillus murinus, Parabacteroides goldsteinii and Shigella dysenteriae). One additional isolate, strain A-C2-0, was a new bacterium. The closest relatives were Bacteroides massiliensis, Bacteroides dorei and Bacteroides vulgatus (< or = 94% similarity). Strain A-C2-0 is a Gram-negative rod that does not form spores and has a G + C content of DNA of 41.5%. Its major cellular fatty acid is C(15:0 ANTEISO), and its major respiratory quinone is MK-9. Cells are aerotolerant but grow only under strict anoxic conditions. They are resistant to cefotaxime and tobramycin. When compared with related Bacteroides spp., the new bacterium was positive for alpha-arabinosidase, negative for glutamyl glutamic acid arylamidase and did not metabolise galactose, glucose, fructose, mannose, raffinose and sucrose. Strain A-C2-0 therefore merits recognition as a member of a novel species within the genus Bacteroides, for which the name Bacteroides sartorii is proposed. The type strain is A-C2-0(T) (= DSM 21941(T) = CCUG 57211(T)).