Bovine Fibroblasts Research Articles

Regulation of fibrillins and modulators of TGFβ in fetal bovine and human ovaries.

Fibrillins 1-3 are stromal extracellular matrix proteins that play important roles in regulating TGFβ activity, which stimulates fibroblasts to proliferate and synthesize collagen. In the developing ovary, the action of stroma is initially necessary for the formation of ovigerous cords and subsequently for the formation of follicles and the surface epithelium of the ovary. FBN3 is highly expressed only in early ovarian development and then it declines. In contrast, FBN1 and 2 are upregulated in later ovarian development. We examined the expression of FBN1-3 in bovine and human fetal ovaries. We used cell dispersion and monolayer culture, cell passaging and tissue culture. Cells were treated with growth factors, hormones or inhibitors to assess the regulation of expression of FBN1-3 When bovine fetal ovarian tissue was cultured, FBN3 expression declined significantly. Treatment with TGFβ-1 increased FBN1 and FBN2 expression in bovine fibroblasts, but did not affect FBN3 expression. Additionally, in cultures of human fetal ovarian fibroblasts (9-17weeks gestational age), the expression of FBN1 and FBN2 increased with passage, whereas FBN3 dramatically decreased. Treatment with activin A and a TGFβ family signaling inhibitor, SB431542, differentially regulated the expression of a range of modulators of TGFβ signaling and of other growth factors in cultured human fetal ovarian fibroblasts suggesting that TGFβ signaling is differentially involved in the regulation of ovarian fibroblasts. Additionally, since the changes in FBN1-3 expression that occur in vitro are those that occur with increasing gestational age in vivo, we suggest that the fetal ovarian fibroblasts mature in vitro.

TGF-β1 promotes bovine mammary fibroblast proliferation through the ERK 1/2 signalling pathway.

The abnormal proliferation of bovine mammary fibroblasts (BMFBs) impairs mammary gland development and lactation. Severe manifestations develop into breast fibrosis, leading to the culling of cows and causing serious losses to the dairy industry. Transforming growth factor β1 (TGF-β1) is an important modulator of cell proliferation and extracellular matrix formation; however, limited information is available on BMFBs. In this study, a convenient and stable culture method for BMFBs was established. Treatment with 5 ng/mL of TGF-β1 significantly promoted the proliferation of BMFBs and accelerated the cell cycle. TGF-β1 stimulation for up to 12 h significantly increased the relative ERK1/2 mRNA expression and enhanced the protein expression of p-ERK1/2 and cyclin D1. Conversely, the ERK1/2 inhibitor PD98059 blocked these TGF-β1 effects. Further exploration using a mouse model showed that TGF-β1 significantly increased the proportion of fibroblasts and accelerating the cell transition from the G1 to G2/M phases. In addition, TGF-β1 enhanced the expression of fibrosis markers, α-SMA and I Collagen, which could be blocked efficiently by the PD98059 in mouse mammary gland. Finally, immunofluorescence analysis confirmed that TGF-β1 promoted fibroblast proliferation in healthy dairy cows after normal long-term dietary corn straw roughage supplementation. It is suggested that the diet may promote mammary fibroblast proliferation by raising the level of TGF-β1. Our study provides new insights into how nutrition causes undesirable changes in mammary gland structure.

Staphylococcus aureus induces TGF-β1 and bFGF expression through the activation of AP-1 and NF-κB transcription factors in bovine mammary gland fibroblasts

Staphylococcus aureus is a common Gram-positive pathogen that causes bovine mastitis, a persistent infection of the bovine mammary gland. To better understand the importance of bovine mammary fibroblasts (BMFBs) and the roles of the TLR–NF-κB and TLR–AP-1 signaling pathways in the regulation of S. aureus-associated mastitis and mammary fibosis, BMFBs cultured in vitro were stimulated with different concentrations of heat-inactivated S. aureus to analyze the gene and protein expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), transforming growth factor beta 1 (TGF-β1), basic fibroblast growth factor (bFGF) as well as the protein expression of nuclear factor-kappa B (NF-κB) and activation protein-1 (AP-1) by means of quantitative polymerase chain reaction (qPCR) and western blotting, respectively. Specific NF-κB and AP-1 inhibitors were also used to investigate their effects on the regulation of TGF-β1 and bFGF expression. The results indicated that, in addition to increasing mRNA and protein expression of TLR2 and TLR4, S. aureus could also upregulate TGF-β1 and bFGF mRNA expression and secretion through the activation of NF-κB and AP-1. The increase in TGF-β1 and bFGF expression was shown to be inhibited by AP-1- and NF-κB-specific inhibitors. Taken together, S. aureus induces TGF-β1 and bFGF expression through the activation of AP-1 and NF-κB in BMFBs. This information offers new potential targets for the treatment of bovine mammary fibrosis.

Sodium humate accelerates cutaneous wound healing by activating TGF-β/Smads signaling pathway in rats

Sodium humate (HA-Na) has been topically used as a wound healing and anti-inflammatory agent in folk medicine. In the present study, HA-Na was investigated for cutaneous wound healing in Sprague–Dawley rats. HA-Na solution (1.0%, w/v) was topically administered to rats undergoing excision wound models. Healing was assessed with a recombinant bovine basic fibroblast growth factor for external use as positive control. Wound healing rates were calculated on Day 3, 6, 9, 14 and 21 after injury, and tissues were also harvested after the same intervals for histological analysis. In addition, tissue hydroxyproline levels were measured. Furthermore, mRNA levels and protein expressions of transforming growth factor-β1, 2, 3 (TGF-β1, 2, 3) were determined by RT-PCR and western blot. Protein expression levels of Smad-2, -3, -4 and -7 were also detected by western blot. Our study demonstrates that HA-Na has the capacity to promote wound healing in rats via accelerated wound contraction and increased hydroxyproline content. More importantly, these wound healing effects of HA-Na might be mediated through the TGF-β/Smad signaling pathway. HA-Na may be an effective agent for enhanced wound healing.

Efficacy of recombinant bovine basic fibroblast growth factor in treatment of dry eye following phacoemulsification with IOL implantation

Background The study on eye surface damage following phacoemulsification with intraocular lens (IOL) implantation is increasingly concerned, and these symptoms were associated with dry eye and often treated by polyethylene glycol eyedrops to remit the discomfortableness. Recombinant bovine basic fibroblast growth factor (rb-bFGF) eyedrops contains neurotrophic factors, but its effect on eye surface damage is worth researching. Objective This study was to evaluate the repair effects of rb-bFGF on ocular surface injury after phacoemulsification with IOL implantation. Methods A randomized controlled trail was designed. Ninety eyes of 72 consecutive patients with age-related cataract were enrolled in Fenyang Hospital of Shanxi Province from September 2010 to August 2013 under the informed consent. Phacoemulsification with IOL implantation was performed on all the eyes, and tobramycin and dexamethasone eye drops was used for 15 days as basis therapy. According to the treatment sequence, the operative eyes were assigned to rb-bFGF eyedrops group, polyethylene glycol eyedrops group and basis therapy group.rb-bFGF drops and polyethylene glycol drops were topically administered 4 times per day since the first day after surgery for consecutive 30 days in corresponding group, and only basis therapy was maintained in the basis therapy grouply. Corneal fluorescence (FL) staining scores, breakup time of tear film (BUT) and Schirmer Ⅰ test (SⅠt) without topical anesthesia were examined in 1 day before operation and 1 day, 7 days, 15 days and 30 days after operation. The efficacy was intergrouply compared. Results No significant differences were seen in the demography and the relevant surface examinational outcomes among the rb-bFGF group, polyethylene glycol drops group and the basis therapy group before surgery (age: F=1.50; gender: χ2=0.336, both at>0.05; FL: F=0.31; BUT: F=0.65; SⅠt: F=0.57; all at P>0.05). Compared with the before operation, FL scores were obviously increased, and BUT values were reduced and SⅠt values were elavated in all the eyes early stage of surgey and then gradually improved with the lapse of postoperative time, showing significant differences (Ftime=7.83, 7.32, 7.17, all at P 0.05). However, there were still significant differences between the before and after operation in the basis were closed to those of before surgery therapy group (all at P<0.05). In addition, significant differences were found in corneal FL scores, BUT and SⅠt among these three groups (Fgroup=5.08, 4.15, 4.61, all at P<0.05). In postoperative 15 days and 30 days, the SⅠt values were (12.32±1.18) and (11.32±1.98) mm/5 min, which were significantly lower than (14.36±1.77) and (13.36±2.32) mm/5 min in the polyethylene glycol drops group and (17.25±2.24) and (13.25±2.53) mm/5 min in the basis therapy group (all at P<0.05). Conclusions The topical application of rb-bFGF combined with tobramycin and dexamethasone eyedrops can improve the dry eye-related symptoms and promote the repair of the ocular surface injury after phacoemulsification with IOL implantation, and the clinical efficacy of rb-bFGF eyedrops is better than that of polyethylene glycol eyedrops or only tobramycin and dexamethasone eyedrops. Key words: [Key words] Dry eye/therapy; Cataract/surgery; Phacoemulsification; Comparative studies; Eyedrops; Recombinant bovine basic fibroblast growth factor; Polyethylene glycol

Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands.

Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription- activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.

Generation of transgenic cattle expressing human β‐defensin 3 as an approach to reducing susceptibility to Mycobacterium bovis infection

Bovine tuberculosis results from infection with Mycobacterium bovis, a member of the Mycobacterium tuberculosis family. Worldwide, M. bovis infections result in economic losses in the livestock industry; cattle production is especially hard-hit by this disease. Generating M. bovis-resistant cattle may potentially mitigate the impact of this disease by reducing M. bovis infections. In this study, we used transgenic somatic cell nuclear transfer to generate cattle expressing the gene encoding human β-defensin 3 (HBD3), which confers resistance to mycobacteria in vitro. We first generated alveolar epithelial cells expressing HBD3 under the control of the bovine MUC1 promoter, and confirmed that these cells secreted HBD3 and possessed anti-mycobacterial capacity. We then generated and identified transgenic cattle by somatic cell nuclear transfer. The cleavage and blastocyst formation rates of genetically modified embryos provided evidence that monoclonal transgenic bovine fetal fibroblast cells have an integral reprogramming ability that is similar to that of normal cells. Five genetically modified cows were generated, and their anti-mycobacterial capacities were evaluated. Alveolar epithelial cells and macrophages from these cattle expressed higher levels of HBD3 protein compared with non-transgenic cells and possessed effective anti-mycobacterial capacity. These results suggest that the overall risk of M. bovis infection in transgenic cattle is efficiently reduced, and support the development of genetically modified animals as an effective tool to reduce M. bovis infection.

Molecular cloning and expression vector construction of bovine TRIM28.

The bovine TRIM28 gene was amplified from ovary tissue by using RT-PCR. The TRIM28 gene was inserted into the eukaryotic expression vector pIRES2-EGFP and transfected into bovine fetal fibroblasts by using Lipofectamine 3000. TRIM28 mRNA and protein were detected by fluorescence microscope and western blotting. The results showed that the full length of TRIM28 was cloned and pIRES2-EGFP-TRIM28 was constructed successfully. EGFP expression was observed, and the pIRES2-EGFP-TRIM28 transfected group expressed more TRIM28 protein than that by the pIRES2-EGFP group. The TIMR28 gene has been successfully transferred into bovine fetal fibroblasts.

Photocurable gelatin hydrogels for cell encapsulation

Event Abstract Back to Event Photocurable gelatin hydrogels for cell encapsulation Timothy Hughes1*, Lingli Li1, 2*, Conglie Lu2*, Mei Chen2*, Huixiang Ma2*, Lei Wang3*, Xiaojuan Hao1*, Keith Mclean1* and Chen Hao2, 3* 1 CSIRO, Manufacturing, Australia 2 Wenzhou Medical University, Wenzhou Eye Hospital, China 3 Wenzhou Institute of Biomaterials and Engineering, China Introduction: Tissue engineering requires the implantation of cells along with a supportive scaffold. As such, degradable support scaffolds that are amenable to minimally invasive implantation are attractive. With this in mind, we have explored the use of phototriggered thiol-ene reactions as a method to produce injectable and photocurable cell supportive scaffolds that can be cured in the presence of cells (Figure 1). Figure 1: Conceptual diagram of in situ cured scaffold in the presence of cells Materials and Methods: Type A Porcine skin (Bloom 100) Gelatin (Sigma Aldrich) was used for experiments. Vinyl functionalised gelatines were produced from gelatine and vinyl anhydrides (eg: acrylate and pentanoic anhydride)[1]. While thiolylated gelatin was prepared from gelatin and cysteine with EDC and NHS[1]. The products were purified by dialysis (1 kDa MWCO) and freeze dried before being reconstituted with saline (0.9 %). Light triggered thiol-ene reactions were used to crosslink the vinyl functionalised gelatins with multifunctional thiols included thiolated gelatin. The rheological properties of the precursor formulations as well as the final gels and their cure profiles were measured using photorheology. The effect of solids content, ratio of components and light intensity were investigated. The water content, optical transparency and mechanical properties and microscopic morphologies of the gels were also measured. The ability of bovine corneal endothelial cells (bCECs) and fibroblast cells (L929) to grow on the gels was investigated. The in situ curing of gels in the presence of living cells was also explored. Finally a gelatin hydrogel was cured in situ to fill corneal defects in rabbits(trephine, 3 mm diameter by 200 μm deep) to assess the corneal biocompatibility of the hydrogels. Results and Discussion: Thiol-ene crosslinked gelatins were able to be cured within 2 min. of exposure to 365 nm light resulting in optically transparent gels with water contents about 80-85 %. The resulting soft gels possessed high viability of both L929 and bCECs (>80 %). The mechanical properties of the gels could be tuned by altering the feed ratio of the components and the light intensity used during the cure. Live/Dead staining confirmed that both bCEC and L929 retained high cell viability in culture following in situ cure of the gelatin hydrogels containing the cells (Figure 2). Moreover, laser confocal imaging indicated that the cells were evenly distributed within the hydrogel. Figure 2: 3D Laser confocal fluorescent imaging of L929 cells pre stained with DilC(3) after 7 days of cell culture in a hydrogel matrix prepared from a formulation of 150 mg/ml 2:1 Gel-LA to Gel-SH + Irgacure 2959 (0.5 %) cured with 100 mW/cm2 for 1min Corneal defects in rabbit corneas were filled with in situ cured gelatine hydrogel and the wounded cornea re-epithelialized in 3 to 7 days with minimal inflammation (Figure 3). Figure 3: Slit lamp microscope images of corneal defects at day 1 (left) and 7 (right) stained with fluorescein filled with gelatin hydrogel matrix prepared from a formulation of 150 mg/ml 2:1 Gel-LA to Gel-SH + Irgacure 2959 (0.5 %) cured with 100 mW/cm2 for 1min Conclusions: Phototriggered thiol-ene reactions are rapid and facile methods of forming crosslinked gelatin hydrogels. They resulted in transparent gels with mechanical properties suitable for soft tissue replacements. In addition, they could be cured in the presence of living cells which maintained high viability for over 7 days post crosslinking. Moreover, the in situ cured gels were able to heal rabbit corneal defects in vivo with low inflammation and rapid re-epithelialization. Therefore, the resulting materials are promising candidates for corneal tissue engineering substrates. This study is supported by Chinese Scholarship Council, the International Scientific and Technological Cooperation Project (No. 2012DFB30020) and High-end Foreign Experts Recruitment Program (China, No.GDW20133300101).

Matrix attachment regions included in a bicistronic vector enhances and stabilizes follistatin gene expressions in both transgenic cells and transgenic mice

In the present study, follistatin (FST) gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region (MAR) were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous FST indicated that the pMAR-CAG-FST-IRES-AcGFP1-polyA-MAR (pMAR-FST) vector had higher capacity to form mono- clonal transgenic cells than the vector without MAR, though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the pMAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous FST was expressed in all of the pMAR- FST transgenic mice at F0 ,F 1 and F2. Total muscle growth in F0 mice was significantly greater than in wild-type mice, with larger muscles in fore and hind limbs of transgenic mice. pMAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle develop- ment and the trend of muscle growth. In conclusion, a pMAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes FST gene expressions in both transfected cells and transgenic mice.

An in vitro model of foreign body giant cell formation mediated by damage-associated molecular patterns

Event Abstract Back to Event An in vitro model of foreign body giant cell formation mediated by damage-associated molecular patterns Jeremy Antonyshyn1, 2 and Lindsay E. Fitzpatrick1 1 Queen's University, Department of Chemical Engineering, Canada 2 University of Toronto, Institute of Biomaterials & Biomedical Engineering, Canada Introduction: Protein adsorption to biomaterials is a well-known phenomenon thought to contribute to the progression of the foreign body reaction (FBR). While the contribution of serum proteins has been studied extensively, those derived from tissues damaged during implantation have received little attention. Toll-like receptors (TLRs) may mount an inflammatory response against these damage-associated molecular patterns (DAMPs) through their activation of NF-κB and AP-1 transcription factors[1]. The purpose of this study was to determine whether TLR-mediated macrophage recognition of DAMPs adsorbed to biomaterial surfaces contributes to the progression of the FBR. Methods: Fetal bovine serum (FBS) and 3T3 fibroblast lysate were used as sources of serum proteins and DAMPs, respectively. Total protein concentrations were determined by microBCA assay. Tissue culture polystyrene (TCPS) was incubated in lysate and/or FBS, then rinsed with phosphate-buffered saline (PBS). Activation of NF-κB/AP-1 in RAW-Blue macrophages induces expression of secreted embryonic alkaline phosphatase (SEAP), the enzymatic activity of which can be quantified with the QUANTI-Blue assay. These cells, cultured in 10% heat-inactivated FBS, were exposed to TCPS alone (negative control), lipopolysaccharide (LPS; positive control), and lysate/FBS-adsorbed TCPS. Culture was ended after 24hrs. Morphology of May-Grünwald-Giemsa –stained cells was evaluated. IL-4 and IFN-γ concentrations were determined by ELISAs. NF-κB/AP-1 –dependent SEAP activity was quantified. All experiments were run in triplicate, ANOVAs with Tukey’s post-hoc analyses used, and p<0.05 accepted as statistically significant. All values reported as mean±SD. Results and Discussion: Lysate induced significantly greater macrophage fusion than TCPS (39.2±13.9% vs 0.5±0.3%, respectively; p<0.05), yielding multinucleated cells (27.0±19.9 nuclei/cell; 9068.0±6548.0µm2) of irregular morphology and random nuclear arrangement (Fig 1). Previous models have relied on IL-4 and IFN-γ to induce fusion[2], but neither was detected suggesting an alternative mechanism. Lysate induced NF-κB/AP-1 activation to the same extent as known TLR-agonist LPS (11.4±0.1 vs 11.9±1.1 fold greater than TCPS, respectively), which was significantly greater than that induced by FBS (0.3±0.1 fold; p<0.001). While this suggests that TLRs mount an inflammatory response against DAMPs but not serum proteins, both compete for adsorption as evidenced by the dose-dependent suppression of NF-κB/AP-1 activity with dilution of lysate in FBS but not PBS (p<0.0001; Fig 2). Importantly, lysate continued to elicit significant activation compared to FBS when comprising as little as 0.06% of total protein added (p<0.05), supporting the relevance of DAMPs to the FBR in vivo. Conclusion: These findings indicate that DAMPs-adsorbed biomaterials elicit an inflammatory response in macrophages and promote their fusion to yield foreign body giant cells, the hallmark of the FBR. Canada Foundation for Innovation’s (CFI) John R. Evans Leaders Fund (JELF); Queen’s University Senate Advisory Research Committee (SARC) Grant

Reversine Increases the Plasticity of Long-Term Cryopreserved Fibroblasts to Multipotent Progenitor Cells through Activation of Oct4.

Reversine, a purine analog, had been evidenced that it could induce dedifferentiation of differentiated cells into multipotent progenitor cells. Here, we showed that reversine could increase the plasticity of long-term cryopreserved bovine fibroblasts, and reversine-treated cells achieved the ability to differentiate into all three germ layers cells, such as osteoblasts and adipocytes from mesoblast, neurocyte from ectoderm, hepatocytes and smooth muscle cells from endoderm. Moreover, treatment of reversine caused the grow arrest of fibroblasts at G2/M and distinct cell swelling resulting in the formation of polyploid cells. In parallel, reversine treatment induced a multipotency of fibroblasts might be attributed to the activation of histone modifications, especially the degression of DNA methylation. However, molecular and cellular experiments suggested that reversine treatment enhanced selectively the expression of pluripotent marker gene Oct4 and mesenchymal marker genes CD29, CD44 and CD73, but Sox2 and Nanog were not detected. Taken together, these results clearly demonstrate the ability of reversine to dedifferentiation of long-term cryopreserved somatic cells through activation of pluripotent gene Oct4.

Establishment of cell-based transposon-mediated transgenesis in cattle

Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the piggyBac (PB) and sleeping beauty (SB) transposon systems were assessed for stable gene transfer into the cattle genome. Bovine fibroblasts were transfected either with a helper-independent PB system or a binary SB system. Both transposons were highly active in bovine cells increasing the efficiency of DNA integration up to 88 times over basal nonfacilitated integrations in a colony formation assay. SB transposase catalyzed multiplex transgene integrations in fibroblast cells transfected with the helper vector and two donor vectors carrying different transgenes (fluorophore and neomycin resistance). Stably transfected fibroblasts were used for SCNT and on in vitro embryo culture, morphologically normal blastocysts that expressed the fluorophore were obtained with both transposon systems. The data indicate that transposition is a feasible approach for genetic engineering in the cattle genome.

242 HIGHLY EFFICIENT SLEEPING BEAUTY TRANSPOSON-MEDIATED TRANSGENESIS IN BOVINE FETAL FIBROBLASTS

Active transposon-mediated transgenesis is an emerging tool for basic and applied research in livestock. We have demonstrated the effectiveness of a helper-independent piggyBac transposon (pGENIE-3) for gene transfer into the genome of bovine cells (Alessio et al. 2014 Reprod. Domest. Anim. 49, 8). Here, we extend our previous research by examining the suitability of a Sleeping Beauty (SB) transposon-based methodology to deliver transgenes into the genome of bovine fetal fibroblasts (BFF), and the ability of these cells to support in vitro embryo development upon somatic cell nuclear transfer (SCNT). In a first experiment, BFF were chemically cotransfected (JetPRIME®, Polyplus-transfection, Illkirch, France) with a helper plasmid (pCMV-SB100X), which carries an expression cassette for the SB transposase, and the donor vector (pT2/Venus/RMCE) harboring an expression cassette for a fluorescent protein (Venus) flanked by the SB inverted terminal repeats (ITR). Three different ratios of helper and donor plasmids were studied: 1 : 2, 1 : 1 and 2 : 1. After 15 days of culture, the number of fluorescent colonies was counted on an inverted microscope. When vectors were used at ratios of 1 : 1 and 2 : 1, a 78-fold and 88-fold increase (P ≤ 0.05) in the number of fluorescent colonies compared with that in the no-transposase control were calculated. In a second experiment, BFF were chemically cotransfected with the helper vector pCMV-SB100X, and 2 donor transposons: pT2/Venus/RMCE and pT2/SV40-Neo. The former harbors a neo resistance cassette framed by SB ITRs. Different ratios of helper:donors (1 : 1 : 1, 2 : 1 : 1 and 2 : 0.5 : 0.5) were studied, and each ratio compared with a no-transposase control. After 15 days of antibiotic selection, the number of G418-resistant colonies was determined. Every time a functional SB transposase vector was included, the number of fluorescent and G418-resistant colonies was markedly higher compared with that in the respective control without transposase (P ≤ 0.001). Interestingly, all G418-resistant colonies expressed Venus. Molecular characterisation of genomic insertions in 6 monoclonal cell lines was performed by PCR and splinkerette PCR. PCR analysis confirmed presence of the Venus transgene in all cell lines. Splinkerette PCR results revealed at least 15 transposase-catalyzed genomic insertions of the transgene. Individual cells from a polyclonal SB transgenic fibroblast culture were used as nuclear donors to produce zona-free SCNT embryos. Of the reconstructed embryos, 33% reached blastocyst stage and about half of them expressed Venus. In conclusion, SB transposase is able to actively transpose monomeric copies of transgenes into the genome of bovine cells, which can be reprogrammed upon nuclear transfer to generate morphologically normal embryos expressing the transgene of interest.

53 TARGETED SCREEN FOR AMINO ACIDS THAT REGULATE BOVINE INNER CELL MASS DEVELOPMENT

Pluripotency relies on species-specific amino acid (AA) metabolism. In the mouse, inner cell mass (ICM) and ICM-derived pluripotent stem cells (PSCs) need threonine, which is catabolized by threonine dehydrogenase (TDH) into acetyl–CoA and glycine. Depleting (Δ) the culture medium of threonine (ΔT) or blocking TDH activity induces PSC death. By contrast, human PSCs do not survive without lysine (ΔK), leucine (ΔL), or methionine (ΔM). Since isolated bovine PSCs cannot be propagated in vitro, we screened for AAs that selectively support pluripotent ICM cells in intact bovine embryos. Five days (D5) post-IVF, embryos were transferred into glutamine-free synthetic oviduct fluid (gSOF) with Eagle’s nonessential (NE) and essential (E) AAs (gSOF_AA) plus BSA. Embryos were individually cultured until D8 under different conditions. Statistical significance was determined using Fisher’s exact test for blastocyst development (morphological grading to IETS standard) and t-tests for cell numbers (differential stain) and gene expression (quantitative or qPCR). Removal of BSA reduced grade 1–3 blastocyst (B1–3) development (37% v. 25%, n = 3; P &lt; 0.001). Depleting NEAAs from gSOF_AA did not significantly decrease B1–3, but depleting all 12 EAAs did (25% v. 8%, n = 6; P &lt; 0.001). Because ΔEAA was most effective, we focused on this. Experiments were conducted in gSOF+NEAA and compared with gSOF_AA as a positive control (n = 2–6 replicates). One (ΔT, ΔM), two (ΔMT, ΔCM, ΔCT; ΔIL, ΔIK, ΔKL), three (ΔCMT, ΔIKL), or six (ΔHPRVWY) EAA drop-out did not affect blastocyst formation, even when NEAAs were also removed for ΔT and ΔM groups (n = 3). However, depleting another six (ΔCIKLMT), nine (+CMT, +IKL), or eleven EAAs (+T, +M) increasingly compromised B1–3 (P &lt; 0.05). Because no clear EAA candidates emerged from the screen, we focused on TDH. TDH mRNA was present at similar levels in microsurgically isolated (by microblade) trophectoderm (TE) and chemically isolated (by Triton X-100) ICM, but undetectable in five adult tissues. Despite ΔT medium showing no effect, exposure to the TDH inhibitor QC1 (50 µM) reduced B1–3 and B1–2 compared with a dimethylsulfoxide (DMSO) solvent control (25% v. 37% and 8% v. 19%, n = 8; P &lt; 0.005). ICM and TE cell numbers were equally reduced in QC1 v. DMSO-treated blastocysts (10 v. 19 and 37 v. 67 with N = 21 and N = 29 embryos, respectively, n = 3; P &lt; 0.005). Yet TDH, hypoblast (PDGRFα), epiblast (NANOG, FGF4, SOX2), and trophoblast (CDX2, KRT8) markers were not consistently affected by QC1. We next applied 3-hydroxynorvaline (3-HNV), which TDH hydrolyses into glycine and propionyl-CoA instead of acetyl-CoA. Compared with solvent controls, 3-HNV (300 µM) killed all embryos and bovine fetal fibroblasts within 3 days in ΔT medium. This toxic effect was fully rescued by &gt;10-fold T-supplementation. Thus, 3-HNV protein incorporation, rather than acetyl-CoA reduction, may nonspecifically impair cellular function. In summary, we found that bovine ICM formation did not specifically depend on metabolizing threonine or any other single EAA. Research was supported by AgResearch Core Funding.

243 EFFICIENT EDITION OF THE BOVINE PRNP PRION GENE IN SOMATIC CELLS AND IVF EMBRYOS USING THE CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 SYSTEM

The rapid introduction of engineered nucleases technologies, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR), provides new opportunities for editing genes in a targeted and rather simple fashion. Few reports are available regarding CRISPR efficiency in domestic species. Here, the CRISPR/Cas9 system was employed to develop knockout and knock-in alleles of the bovine PRNP gene, responsible for bovine spongiform encephalopathy (mad cow disease), both in bovine fetal fibroblasts and in IVF embryos. Five sgRNAs were designed to target a 875-bp region within prnp exon 3; all 5 were co-delivered with hCas9 and a homologous recombination vector carrying gfp (pHRegfp). For cells, 3 transfection conditions were compared: 2 μg of hCas9 + 1 μg of sgRNAs mix ± 2 μg pHREGFP (1X) versus 4 μg of hCas9 + 2 μg of sgRNAs mix ± 4 μg of pHREGFP (2X). For IVF zygotes, cytoplasmic injection was conducted with 2 RNA concentrations: (a) 50 ng μL–1 hCas9 RNA + 25 ng μL–1 sgRNAs mix (RNA1X), ±50 ng μL–1 pHREGFP, and (b) 100 ng μL–1 hCas9 + 50 ng μL–1 sgRNAs mix (RNA2X), ±100 ng μL–1 pHREGFP, which were compared with plasmid injections with 100 ng μL–1 pCMVCas9 + 50 ng μL–1 pU6sgRNAs mix (DNA2X), ±100 ng μL–1 pHREGFP. The pHREGFP was always injected as plasmid, under the same conditions as hCas9. DNA from cells was subjected to PCR, Surveyor assay, and sequence analysis. Embryo analysis was conducted on whole-genome-amplified DNA from blastocysts, followed by PCR assays and sequencing. In cells, 2X transfection resulted in indels and amplification of PCR products of lower MW than the wild-type, indicative of the deletion of a part of the targeted PRNP region. However, it was not possible to detect an effect for 1X transfection. For the group transfected with pHREGFP, insertion of a partial EGFP sequence was detected (383 bp). Regarding embryo injection, higher blastocyst rates were obtained in all groups injected with RNA (Table 1). In 48% (21/43) of the sequenced blastocysts specific gene editing was detected (Table 1). Modifications varied among single base pair shift (3/43; 7%), high level of mismatches all over the targeted sequence and vicinity (12/43; 27.9%), full deletion of the 875-bp region (1/43; 2.3%), and partial insertion of 100–498 bp pHREGFP fragments between the HR arms (5/24; 20.8%). Most of these modifications occurred in a mosaic fashion (76%). Results demonstrate that CRISPR/Cas can be efficiently applied for site-specific edition of domestic species genomes. Table 1.In vitro development and gene editing efficiency of embryos injected with plasmids or RNA coding for CRISPR/Cas9 system targeting PRNP

Improved cellular thermotolerance in cloned Holstein cattle derived with cytoplasts from a thermotolerant breed

The objective of this study was to compare the thermotolerance of ear fibroblasts derived from various SCNT cattle. Specimens were produced from cloned embryos that had been reconstructed using donor cells (d) from the same Holstein cow (Hd) and the ooplasm (o) from Holstein cattle (Ho) or Taiwan yellow cattle (Yo). Polymorphism in the D-loop region of mitochondrial DNA in ear fibroblasts derived from SCNT cattle reconstructed with the Y ooplasm and H donor cells (SCNT-Yo-Hd) indicates that the cytoplasm originated from Bos indicus. The rates of apoptosis in heat-shocked ear fibroblasts derived from SCNT-Yo-Hd cattle (1.9%) and purebred Y cattle (1.5%) were significantly (P < 0.05) lower than those of cells derived from SCNT cattle reconstructed with the H ooplasm (SCNT-Ho-Hd: 3.4%), donor cells (4.0%), and purebred Holstein (4.1%) cattle. At the protein level, the relative abundances of apoptosis-inducing factor, B cell lymphoma 2–associated X protein, endonuclease G, cytochrome c, cysteinyl aspartate-specific proteinases 3, 8 and 9 in ear fibroblasts derived from SCNT-Yo-Hd cattle were significantly (P < 0.05) lower than those of cells derived from SCNT-Ho-Hd cattle after heat shock. In contrast, the relative abundances of heat shock proteins 27, 70 and B cell lymphoma 2 in ear fibroblasts derived from SCNT-Yo-Hd cattle were higher (P < 0.05) than those of fibroblasts derived from SCNT-Ho-Hd cattle. Moreover, heat-shocked ear fibroblasts derived from SCNT-Yo-Hd cattle have a significantly (P < 0.05) lower percentage of apoptosis-inducing factor–positive nuclei than do heat-shocked ear fibroblasts derived from SCNT-Ho-Hd cattle (11.1% vs. 18.5%). Taken together, these results report that ear fibroblasts derived from SCNT cattle reconstructed using the Y ooplasm are more thermotolerant than ear fibroblasts derived from SCNT cattle reconstructed using the H ooplasm. This is an indication that the cytoplasm may be a major determinant of thermal sensitivity in bovine ear fibroblasts.

Effect of functional groups on physicochemical and mechanical behavior of biocompatible macroporous hydrogels

The increasing interest in studying the properties of biocompatible hydrogels is due to their possible applications in bioengineering. Properties of hydrogels based on N-isopropylacrylamide (NIPAM) and the effect caused by copolymerization with 2-acrylamido-2-methylpropanesulfonic acid (AMPS) or N-acryloyl-tris-(hydroxymethyl)aminomethane (HMA) were investigated. Hydrogels were synthesized by free radical polymerization at room temperature or by cryogelation at −18°C. The presence of different functional groups (isopropyl, −SO3−, and –OH) and thermal conditions of polymerization affected the morphology and physicochemical and mechanical properties of hydrogels. Scanning electron microscopy (SEM) revealed the presence of macropores created by cryogelation with the morphology of the pores dependent on chemical composition of the copolymer. Poly(NIPAM-co-HMA) has spherical and isolated pores, whereas PNIPAM and Poly(NIPAM-co-AMPS) showed ellipsoidal interconnected pores. Three different water states were detected by differential scanning calorimetry (DSC), indicating the presence of nano- and macropores. Elastic modulus (E) was measured to be around 3–6.5kPa by uniaxial compression. However, higher E values (20–30kPa) and an anisotropic mechanical response were observed for PNIPAM and PNIPAM-co-AMPS hydrogels with ellipsoidal pores, a behavior that is almost similar to that of cartilage and bone tissues. Cytocompatibility studies using bovine fibroblasts (BFs) indicated good cell attachment and proliferation on PNIPAM-based hydrogel surfaces, although initially the cell adhesion varied depending on the composition of the surface. These hydrogels could be an interesting choice for the development of scaffolds in tissue engineering.

BEFS-PPARγ2 cells incubated with trans-11 C18:1 exhibit more beneficial fatty acid synthesis

Responses of the PPARγ2 (an adipogenic lineage determinant) over-expressed immortalized bovine embryonic fibroblast cell line (BEFS-PPARγ2 cells) to trans-11 C18:1 (TVA) incubation were evaluated. BEFS-PPARγ2 cells were well differentiated. Stearoyl-CoA desaturase 1, an endoplasmic reticulum enzyme that can catalyze conversion of TVA into cis-9, trans-11 CLA, exhibited a significantly (p<0.05) higher mRNA expression after 4 h, compared with controls. Enzyme activity peaked, and was significantly (p<0.05) higher after 6 h, compared with controls. With extension of the incubation time, TVA absorption into cells was significantly (p<0.05) higher with significantly (p<0.05) more conversion into cis-9, trans-11 CLA, compared with shorter incubation times. TVA incubated cells exhibited significantly (p<0.05) higher levels of arachidonic, eicosapentaenoic, and docosahexaenoic acids than controls. BEFS-PPARγ2 cells can be used as a model system and TVA incubation is a good approach for research into cis-9, trans-11 CLA-fortified beef production.

Effectiveness of non-biodegradable poly(2-hydroxyethyl methacrylate)-based hydrogel particles as a fibroblast growth factor-2 releasing carrier

ObjectivesDental resin-based restorative materials are used in a variety of dental treatment modalities such as root-end filling, perforation sealing, and adhesion of fractured roots. However, the prognosis after such treatments is not necessarily favorable because they fail to promote healing of the surrounding alveolar tissue. In the present study, non-biodegradable poly-2-hydroxyethyl methacrylate (polyHEMA)-based hydrogel particles were fabricated as a carrier vehicle for drug delivery that is applied to dental resins. MethodsThe loading and release characteristics of bovine serum albumin (BSA) and fibroblast growth factor-2 (FGF-2) from the polyHEMA-based hydrogel particles were evaluated over time in culture. The hydrogel particles were immersed into an aqueous FITC-labeled BSA solution and were observed using confocal laser scanning microscopy (CLSM). To determine the activity of the FGF-2 released from the particles, the proliferation of osteoblast-like cells cultured with eluates collected from the particles for up to 14 days was determined. ResultsCLSM revealed that BSA was adsorbed to the surface of the hydrogel particles. A sustained release of BSA and FGF-2 from the particles was detected for up to 14 days. The eluates from the FGF-2-loaded particles increased the proliferation of the osteoblast-like cells, suggesting that the activity of FGF-2 was maintained for at least 2 weeks within the particles. SignificanceThese polyHEMA-based non-degradable hydrogel particles may be useful tools that can be applied to dental restorative materials to achieve sustained delivery of drugs that promote tissue regeneration.