A Cell’s Viscoelasticity Measurement Method Based on the Spheroidization Process of Non-Spherical Shaped Cell

The mechanical properties of biological cells, especially the elastic modulus and viscosity of cells, have been identified to reflect cell viability and cell states. The existing measuring techniques need additional equipment or operation condition. This paper presents a cell’s viscoelasticity measurement method based on the spheroidization process of non-spherical shaped cell. The viscoelasticity of porcine fetal fibroblast was measured. Firstly, we introduced the process of recording the spheroidization process of porcine fetal fibroblast. Secondly, we built the viscoelastic model for simulating a cell’s spheroidization process. Then, we simulated the spheroidization process of porcine fetal fibroblast and got the simulated spheroidization process. By identifying the parameters in the viscoelastic model, we got the elasticity (500 Pa) and viscosity (10 Pa·s) of porcine fetal fibroblast. The results showed that the magnitude of the elasticity and viscosity were in agreement with those measured by traditional method. To verify the accuracy of the proposed method, we imitated the spheroidization process with silicone oil, a kind of viscous and uniform liquid with determined viscosity. We did the silicone oil’s spheroidization experiment and simulated this process. The simulation results also fitted the experimental results well.

36 TREATMENT OF PORCINE FETAL FIBROBLASTS WITH DNA METHYLATION INHIBITORS OR HISTONE DEACETYLATION INHIBITORS IMPROVES DEVELOPMENT OF NUCLEAR TRANSFER EMBRYOS

Epigenetic status of the genome of a donor nucleus has an important effect on the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). DNA methylation inhibitors [such as 5-aza-2′-deoxyctidine (5-aza-dC), zebularine, and RG108] and histone deacetylase inhibitors [such as trichostatin A (TSA), sodium butyrate (NaBu), and scriptaid (SCR)] have been widely used for the alteration of the levels of the epigenetic modification of somatic cells. This study was designed to investigate the DNA methylation status of porcine fetal fibroblast cells treated with TSA or 5-aza-dC and to determine whether treatments with DNA methylation inhibitors or histone deacetylation inhibitors could improve the in vitro development of porcine reconstructed embryos. When the levels of DNA methylation in the PRE-1 sequence (repeat sequence in a euchromatic region) were examined by bisulfite sequencing following treatment of porcine fetal fibroblast cells with TSA or 5-aza-dC for 1 h, DNA methylation was decreased in 5-nm or 50-nm concentrations even if they were not significantly different. To evaluate the effect of DNA methylation inhibitors and histone deacetylation inhibitors on development of porcine nuclear transfer embryos, porcine fetal fibroblast cells were treated with 5 nm of 5-aza-dC, zebularine, or RG108 for 1 h, or with 50 nm of TSA, NaBu, or SCR for 1 h, or treated with both 50 nm TSA and 5 nm 5-aza-dC for 1 h before NT. The reconstructed embryos were electrically fused and cultured in PZM-3 for 6 days. Developmental rates of the reconstructed embryos from donor cells treated with 5-aza-dC, zebularine, or RG-108 to blastocysts significantly increased compared to the control group (21.4, 23.3, and 22.1 v. 12.3%). Blastocyst rates of the reconstructed embryos from donor cells treated with TSA, SCR, and NaBu also were significantly improved compared to the control group (30.0, 23.9, and 22.4 v. 14.5%), and TSA treatment was the highest in blastocyst rates among the treated groups. However, the development rate to the blastocyst stage was not affected when the combination of TSA and 5-aza-dC was treated. In conclusion, treatment of donor cells with DNA methylation inhibitors or histone deacetylase inhibitors improved the subsequent blastocyst development of porcine reconstructed embryos even though combined treatment with both inhibitors had no beneficial effect.

20 PRODUCTION OF NUCLEAR TRANSFER EMBRYOS FROM PORCINE FETAL FIBROBLAST CELLS CARRYING A TETRACYCLINE-INDUCIBLE TRANSGENE

Constitutive expression of A transgene often results in serious physiological disturbances in transgenic animals. For instance, systemic overexpression of human growth hormone in transgenic pigs has resulted in detrimental side effects in general health and reproductive performance. One of the solutions to such problem would be inducible expression of a transgene that may restrict production of foreign proteins from transgenic animals only when needed. In this study, a retrovirus vector was designed to express the green fluorescent protein (GFP) gene under the control of the tetracycline-inducible promoter. Transformation of porcine fetal fibroblast cells was achieved by infection of the cells with the vector and subsequent antibiotic selection. To induce transgene expression, transformed porcine fetal fibroblast cells were cultured in medium supplemented with doxycycline for 48 h. Induction of the GFP gene was verified by the emission of fluorescence from transformed cells. Nuclei of transformed cells with or without doxycycline treatment were transferred into enucleated oocytes, and the induction efficiency was analyzed by monitoring fluorescent emission during development of reconstituted embryos to the blastocyst stage. In addition, differences in the rates of blastocyst development between experimental groups were analyzed by Student's t-test. Blastocyst formation of nuclear transfer embryos using transformed cells with tetracycline-inducible retrovirus vector (12.0%, 128/1072) was not significantly different (P > 0.05) from that with non-inducible control vectors (13.7%, 41/300), suggesting that an introduction of tetracycline-inducible retrovirus vector was not particularly harmful to the development of nuclear transfer embryos. Also, the blastocyst development rate of nuclear transfer embryos after induction of transgene by doxycycline (12.1%, 99/815) was not significantly different (P > 0.05) from that of the non-induced counterparts (11.3%, 29/257), suggesting that the induction of transgene did not affect the development of transgenic clone embryos. In a majority of embryos, high expression of the GFP gene was observed in cloned embryos with transgene induction, whereas poor or no GFP expression was detected in non-induced controls. The results from this study suggest that tetracycline-inducible expression of transgenes in nuclear transfer embryos may be used for production of foreign proteins in transgenic animals in a more controlled manner than with conventional procedures. Further experiments on transfer of cloned embryos carrying such an inducible transgene to recipients may enable production of transgenic pigs with fewer side effects from unregulated expression of the transgene.