Physiology & Growth Biology Research
2006
RNA interference of mouse CFTR function; towards a pig model of cystic fibrosis
D.F. Carlson, A. Geurts, M. Palmer-Densmore,
C.W. Park,
W. Warwick, C. Steer, C. Milla, S. O’Grady,
D. Largaespad, and
S.C. Fahrenkrug
University of Minnesota, Twin Cities
Cystic fibrosis (CF) is the most common life-shortening disease in Caucasians, affecting between 1 in 2,000 and 1 in 4,500 individuals. The gene involved in CF, the transmembrane conductance regulator (CFTR), was identified in 1989. Although mouse models of CF have been generated which manifest some of the electrophysiologic characteristics of CF, their benign pulmonary phenotype renders these models incomplete. We have undertaken the development of a porcine model of CF by RNA interference. As proof of principle, mice were created using a Sleeping Beauty transposon driving the expression of a short hairpin RNA directed against the mouse CFTR mRNA (shCFTR). Twenty-two lines of founder mice were generated by pronuclear injection with methylated or unmethylated transposons and mRNA encoding the SB transposase. Mouse transgenesis was extremely efficient using the methylated transposon, and was accompanied by erasure of transgene methylation in both F0 and F1 animals. Mice were demonstrated to express shCFTR at heritable levels dependent on line in a manner consistent with in vivo methylation of the transgene. Several F1 animals, but not F0 founders, displayed phenotypes consistent with CF, including impairment of chloride transport in colonic epithelia and impairment of reproduction. The abundance of CFTR mRNA and protein in shCFTR expressing mouse epithelial cell lines (HT11, IMCD3) and transgenic mice are currently under investigation and will be presented. The efficacy of this approach has also been investigated in pig cells. We have demonstrated that porcine shCFTR expression results in 90-94% knockdown in a cultured pig endometrial epithelial cells, which also displayed a quantitative loss of CFTR function based on apical membrane Cl- current. Sleeping Beauty was also used to generate a collection of pig fibroblast clones that were characterized for shRNA expression by quantitative PCR. Clonal lines expressing high levels of shRNA’s were identified and provided to the National Swine Center for the generation of knockdown pigs by somatic cell nuclear transfer (cloning).
Development of an in vitro cell culture system for Lawsonia intracellularis
J. Lan Chun, C. Gebhart, and D. Foster
University of Minnesota, St. Paul
Lawsonia intracellularis was only formally identified in 1995. This organism has been shown to be difficult to propagate due to adaption to in vitro growth conditions. This limitation is a problem for understanding the pathogenesis of Lawsonia intracellularis. For a more effective study of Lawsonia intracellularis, development of in vitro cell culture system is required to compare the more limited in vivo analysis. Porcine endometrial glandular epithelial (PEGE) and porcine embryonic fibroblast (PEF) cells were selected for propagation. Cells were seeded in tissue culture flasks with no antibiotics at about 10~15% confluence, and were incubated for 24hr at 37°C, 5% CO2. After pre-incubation, Lawsonia intracellularis was inoculated into the tissue culture flasks, followed by an induction of a microaerobic environment with hydrogen. Cells infected by Lawsonia bacteria were incubated 5~7 days post-infection at 37°C, in a gaseous environment of 8.0% O2, 8.8% CO2, and 83.2% N2. Detection of Lawsonia bacteria in cultured pig cells was performed using the monoclonal antibody, IPX. In conclusion, PEG and PEF cells showed potential for a new in vitro cell culture system for Lawsonia intracellularis.
Enzymatic manipulation of the pig genome with transposons and recombinases
K.J. Clark, D.F. Carlson, L. Foster, and S.C. Fahrenkrug
University of Minnesota, St. Paul
Recent developments in livestock transgenesis, somatic cell nuclear transfer (SCNT, cloning), and stem cell biology have fueled plans to engineer the pig genome for both agricultural and emerging medical markets. With tissue size and physiology closely resembling humans, pigs can provide excellent large animal models of human disease and gene therapy. An abundant supply of pigs could provide an unlimited resource for organ and cellular xenotransplantation. These diverse applications would benefit greatly from the development of technologies for efficient porcine genetic engineering. We are investigating the use of three vertebrate transposons, Sleeping Beauty (SB), Tol2, and PPTN for porcine cell transgenesis. The PPTN transposon is a novel TC1-family transposon from Plaice (Pleuronectes platessa). Comparison of cloned PPTN transposons reveals 99.5% sequence conservation at the nucleotide level, suggesting a recent colonization of the Plaice genome. The full-length transposase is 50% (67%) and 53% (72%) identical (similar) to SB and Frog Prince, respectively. We have demonstrated the ability of PPTN to function in HT1080 (human) cells, and have examined the efficiency of SB, Tol2, and PPTN function in porcine cells. Efforts to engineer the pig genome would also benefit from site specific genome recombination, enabling selection cassette recycling, and gene activation or silencing. We have therefore studied the ability of CRE and FLP recombinases to mediate genomic rearrangements in pig cells. A positive/negative selection strategy was used to determine the efficiency of CRE and FLP mediated recombination of genome-resident transgenes in pig cells. In addition, a CRE-activated GFP expression system was developed to monitor rates of recombination in pig cell populations. Given their ability to function in porcine cells, transposons and recombinases should be useful tools for genetic engineering for both agricultural and biomedical swine applications.
Insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 mediate TGF β-and myostatin-induced suppression of proliferation in porcine embryonic myogenic cell cultures
E. Kamanga-Sollo, M.S. Pampusch, M.E. White, M.R. Hathaway, amd W.R. Dayton
University of Minnesota, St. Paul
TGF-β superfamily members myostatin and TGF-β1 have been shown to suppress both proliferation and differentiation of myogenic cells. Treatment of cultured porcine embryonic myogenic cells (PEMC) with either TGF- β1 or myostatin increases levels of insulin-like growth factor (IGFBP)-3 and -5 mRNA and protein. Additionally, both IGFBP-3 and IGFBP-5 cause IGF-independent suppression of proliferation in PEMC cultures. Consequently, we have examined the role of these IGFBPs in the ability of TGF- β1 and myostatin to suppress proliferation and differentiation of cultured PEMC. Treatment of PEMC cultures with either myostatin or TGF- β1 significantly (P<0.01) increases levels of both IGFBP-5 and IGFBP-3 mRNA. We have previously shown that immunoneutralization of IGFBP-3 decreases the proliferation-suppressing activity of TGF- β1 and myostatin. Similarly, immunoneutralization of IGFBP-5 also significantly (P<0.05) decreases the proliferation suppressing activity of these molecules. Simultaneous immunoneutralization of both IGFBP-3 and IGFBP-5 in TGF- β1 or myostatin treated PEMC cultures restores both IGF-I and Long-R3-IGF-I-stimulated proliferation rates to 90% of the levels observed in control cultures receiving no TGF- β1 or myostatin treatment (P<0.05). Even though immunoneutralization of IGFBP-3 and -5 increased proliferation rates in TGF- β1 or myostatin-treated PEMC cultures, phosphosmad 2 levels in these cultures were not affected. Consequently, we believe that our data strongly indicate that IGFBP-3 and IGFBP-5 mediate TGF-β1 and myostatin-induced suppression of PEMC proliferation via IGF-independent mechanisms that do not involve phosphosmad 2 signaling.
IGF-I mRNA levels in bovine satellite cell cultures: Effects of fusion and anabolic steroid treatment
E. Kamanga-Sollo, M.S. Pampusch, G. Xi, M.E. White, M.R. Hathaway, and W.R. Dayton
University of Minnesota, St. Paul
Androgenic and estrogenic steroids enhance muscle growth in a number of species; however, the mechanism by which anabolic steroids enhance muscle growth is not known. Castrated male cattle (steers) provide a particularly good model system in which to study the effects of anabolic steroids on muscle growth because they respond dramatically to treatment with both estrogens and androgens. The goal of this study was to determine if treatment of bovine satellite cell (BSC) cultures with 17ß-estradiol (E2) or trenbolone (a synthetic androgen) directly affects proliferation rate or level of mRNA for estrogen receptor (ER)-α, androgen receptor, and growth factors that have been shown to affect muscle growth (insulin-like growth factor (IGF-I), insulin-like growth factor binding protein (IGFBP)-3, and myostatin). BSC cultures were established from the semimembranosus muscles of steers and then treated for 48 hours with various concentrations E2 or trenbolone ranging from 0.001 nM to 10 nM. IGF-I mRNA levels in proliferating BSC cultures were significantly increased at 0.01 (1.9 times controls values, P<0.02) and at 0.1, 1, and 10 nM E2 (2.9, 3.5 and 3.5 times control values, respectively, P<0.0001). Additionally, both 1 and 10 nM trenbolone increased IGF-I mRNA levels to 1.7 times control values (P<0.02). ER-α mRNA was detectable in BSC cultures, and levels were increased (2.3 times control levels, P<0.001) in cultures treated with 0.001 nM E2 but not in cultures treated with higher concentrations of E2. Androgen receptor mRNA levels also were increased (1.5 times control levels, P<0.02) in cultures treated with 0.001 nM trenbolone but not by treatment with higher concentrations of trenbolone. Levels of IGFBP-3 were increased (1.4 times control values, P<0.02) by treatment with 0.001 nM E2 but not by treatment with high concentrations of E2. Myostatin mRNA levels were not affected by any concentration of either of the steroids. Although, levels of IGF-I mRNA were 10 times greater (P<0.02) in fused BSC cultures than in proliferating cultures, treatment of fused cultures for 48 hours with 10 nM E2 increased IGF-I mRNA levels (2.5 times control levels, P<0.02). Both E2 and trenbolone increased 3H-thymidine incorporation rate (1.5 times control levels, P<0.001) in BSC cultures in media containing serum from which IGFBP-3 had been removed by anti-IGFBP-3 affinity chromatography. In summary, treatment of BSC cultures with either E2 or trenbolone increased IGF-I mRNA level and proliferation rate, thus, establishing that these steroids have direct anabolic effects on cells present in the BSC culture.
Map kinase phosphatase-3 regulation in colorectal carcinoma
N. Okoye, M.R. Zillhardt, N. Zeliadt, E. Wattenberg, and L.J. Mauro
University of Minnesota, Twin Cities
Colon cancer is the third most common type of cancer affecting both men and women in the United States and has claimed the lives of an estimated 56,000 people in 2005 alone. The progression of colorectal cancer is due to accumulation of epigenetic and genetic alterations that often include the mutagenesis of the K-ras oncogene. This mutation causes constitutive activation of K-ras and downstream Ras/Raf/MAPK pathway leading to abnormal cell growth. We are interested in how these cells control this pathway to prevent cancer progression. The goals of my studies are: (1) to determine if a negative regulator of the MAPK pathway known as MAP kinase phosphatase-3 (MKP-3) is expressed in colon adenocarcinoma cells; and (2) if expressed, to determine if the expression of MKP-3 is modulated to control the constitutive activation of this pathway. Colon adenocarcinoma cells were treated with mitogens and analyzed to determine MKP-3 mRNA and protein expression and the phosphorylation (pERK) and activation of ERK kinase. High MKP-3 expression is observed in these cells and is uniquely regulated in relation to activated pERK levels following mitogenic stimulation of the pathway. To verify whether MKP3 is modulating ERK activation, future studies will be conducted to 'silence' MKP3 expression and observe how knocking this gene out will affect regulation of the MAPK pathway.
Regulation of CCL2 in a novel fibrosarcoma-bone co-culture model
K.R. Schiller, M.R. Zillhardt, A.J. Beitz, and L.J. Mauro
University of Minnesota, Twin Cities
To understand the aspects of the microenvironment important in metastatic cancers, factors secreted by the bone and tumor must be identified and their role in tumor progression and bone metabolism established. Present cancer models are limited by lack of tissue architecture, absence of multiple cell types and inability to access the site of tumor-bone interactions. We have developed an alternative murine co-culture model using whole neonatal femurs (BN) and the NCTC 2472 fibrosarcoma cell line (FS), capable of in vivo tumor formation in bone. Viability and apoptosis assays indicate both the BN and the FS are viable in these cultures for 2-4 days depending on media conditions. No loss of viability or increased apoptosis is observed in the cartilaginous epiphyses of the bone through day 4. Evidence of apotosis (3.5 fold) was observed in the marrow cavity of bones after 48 hrs in culture as compared to corresponding fresh-dissected BN. FS cells exhibited robust proliferation as assessed by increasing cell numbers throughout the culture period. The secretion profile of several tumor and bone factors was analyzed in conditioned media from these cultures by ELISA. Most significant was the apparent synergy observed in secretion of the chemokine CCL2. Both BN and FS secrete CCL2 at days 1 (BN: 648.5 ± 63.7pg/ml; FS: 1112.4 ± 23.2) and 4 (BN: 3539.3 ± 415.3; FS: 3125.2 ± 523.0) when cultured alone, whereas in co-culture, a greater than additive CCL2 secretion is observed (day 1: 2223.1 ± 61.7; day 4: 14408.7 ± 1441.7). When corrected for proliferation of FS during culture, a 1.5-2.5 fold enhancement in secretion of CCL2 is evident compared to tissue/cell component alone. This co-culture effect was not observed for other factors examined. The concentration of transforming growth factor-ß1 (TGF-ß1) was similar in BN alone (811.6 ± 39.5 pg/ml) and BN-FS co-culture (785.3 ± 20.7) with FS secreting little TGF-ß1 in comparison. Likewise, detectable secretion of matrix metalloproteinase-2 (MMP-2) was only observed in BN alone (21.8 ± 1.6 ng/ml) and co-cultures (18.3 ± 1.4). Therefore, the majority of TGF-ß1 and MMP-2 is secreted by the bone explant. Additional analyses showed no detectable secretion of tumor necrosis factor-a in these cultures. These results indicate this model is viable and that specific components of this simulated microenvironment secrete factors known to influence tumor progression. The interaction of FS with the 3-dimensional bone explants results in the unique regulation of CCL2 secretion, suggesting this model could be a valuable tool to study the bone-tumor microenvironment. Future studies are being conducted to identify the mechanisms of CCL2 regulation in fibrosarcoma and explore the use of this model for study of other metastatic cancers of bone.
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