Primary Fetal Fibroblasts Research Articles

The third helix of the murine Hoxc8 homeodomain facilitates protein transduction in mammalian cells

Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191–206, 2.23 kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminal of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1 μM, and appeared to plateau above a concentration of 1 μM. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500 nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins.

313. AAV-Mediated Disruption of the CFTR Gene in Primary Fetal Fibroblasts To Create a CFTR Knockout Ferret by Nuclear Transfer Cloning

313. AAV-Mediated Disruption of the CFTR Gene in Primary Fetal Fibroblasts To Create a CFTR Knockout Ferret by Nuclear Transfer Cloning

Prostaglandin E2 Inhibits Specific Lung Fibroblast Functions via Selective Actions of PKA and Epac-1

Via their capacities for proliferation and synthesis of matrix proteins such as collagen, fibroblasts are key effectors in the pathogenesis of fibrotic disorders such as idiopathic pulmonary fibrosis. Prostaglandin E(2) (PGE(2)) potently inhibits these functions in lung fibroblasts through receptor ligation and production of the second messenger cAMP, but the downstream pathways mediating such actions have not been fully characterized. We sought to investigate the roles of the cAMP effectors protein kinase A (PKA) and exchange protein activated by cAMP-1 (Epac-1) in modulating these two functions in primary human fetal lung IMR-90 fibroblasts. The specific roles of these two effector pathways were examined by treating cells with PKA-specific (6-bnz-cAMP) and Epac-specific (8-pCPT-2'-O-Me-cAMP) agonists, inhibiting PKA with the inhibitor KT 5720, overexpressing the PKA catalytic subunit, and silencing Epac-1 using short hairpin RNA. PGE(2) inhibition of collagen I expression was mediated exclusively by activation of PKA, while inhibition of fibroblast proliferation was mediated exclusively by activation of Epac-1. PGE(2) and Epac-1 inhibited cell proliferation through activation of the small GTPase Rap1, since decreasing Rap1 activity by transfection with Rap1GAP or the dominant-negative Rap1N17 prevented, and transfection with the constitutively active Rap1V12 mimicked, the anti-proliferative effects of PGE(2). On the other hand, PKA inhibition of collagen was dependent on inhibition of protein kinase C-delta. The selective use of PKA and Epac-1 pathways to inhibit distinct aspects of fibroblast activation illustrate the pleiotropic ability of PGE(2) to inhibit diverse fibroblast functions.

Analysis of gene expression in fetal and adult cells infected with rubella virus

Congenital infection with rubella virus (RUB) leads to persistent infection and congenital defects and we showed previously that primary human fetal fibroblasts did not undergo apoptosis when infected with RUB, which could promote fetal virus persistence [Adamo, P., Asís, L., Silveyra, P., Cuffini, C., Pedranti, M., Zapata, M., 2004. Rubella virus does not induce apoptosis in primary human embryo fibroblasts cultures: a possible way of viral persistence in congenital infection. Viral Immunol. 17, 87–100]. To extend this observation, gene chip analysis was performed on a line of primary human fetal fibroblasts (10 weeks gestation) and a line of human adult lung fibroblasts (which underwent apoptosis in response to RUB infection) to compare gene expression in infected and uninfected cells. A total of 632 and 516 genes were upregulated or downregulated in the infected fetal and adult cells respectively in comparison to uninfected cells, however only 52 genes were regulated in both cell types. Although the regulated genes were different, across functional gene categories the patterns of gene regulation were similar. In general, regulation of pro- and anti-apoptotic genes following infection appeared to favor apoptosis in the adult cells and lack of apoptosis in the fetal cells, however there was a greater relative expression of anti-apoptotic genes and reduced expression of pro-apoptotic genes in uninfected fetal cells versus uninfected adult cells and thus the lack of apoptosis in fetal cells following RUB infection was also due to the prevailing background of gene expression that is antagonistic to apoptosis. In support of this hypothesis, it was found that of a battery of five chemicals known to induce apoptosis, two induced apoptosis in the adult cells, but not in fetal cells, and two induced apoptosis more rapidly in the adult cells than in fetal cells (the fifth did not induce apoptosis in either). A robust interferon-stimulated gene response was induced following infection of both fetal and adult cells and many of the genes upregulated in both cell types were those involved in establishment of an antiviral state; this is the first demonstration of an interferon response at this early stage of human embryonic development. In both fetal and adult cells, interferon controlled but did not eliminate virus spread and apoptosis was not induced in infected fetal cells in the absence of interferon. In addition to the interferon response, chemokines were induced in both infected fetal and adult cells. Thus, it is possible that fetal damage following congenital RUB infection, which involves cell proliferation and differentiation, could be due to induction of the innate immune response as well as frank virus infection.

Wnt-4 Expression Is Increased in Fibroblasts after TGF-??1 Stimulation and during Fetal and Postnatal Wound Repair

Wnt-4 is a mitogen expressed during postnatal repair and scar formation; however, its expression profile during scarless repair is unknown. Transforming growth factor (TGF)-beta1 has high expression during healing with scar formation. Whether TGF-beta1 directly influences Wnt-4 expression in fetal or postnatal fibroblasts has not been examined. Primary fetal and postnatal mouse fibroblasts were stimulated with TGF-beta1 and Wnt-4 expression quantitated by real-time polymerase chain reaction. Fetal E17 and postnatal mouse excisional wounds were also analyzed for Wnt-4 expression by real-time polymerase chain reaction. In E17 fibroblasts after TGF-beta1 stimulation, Wnt-4 expression increased 4-fold at 1 hour (p < 0.05) and peaked with an 11-fold increase at 2 hours (p < 0.05). By 24 hours, expression decreased to 2-fold baseline levels (p < 0.05). In postnatal fibroblasts, Wnt-4 expression also increased after TGF-beta stimulation, but peak expression was larger and relatively delayed, with a 17-fold increase at 12 hours (p < 0.005). Expression levels at 24 hours were still 4-fold greater than baseline (p < 0.05). In E17 fetal skin, Wnt-4 expression was 3.5-fold greater compared with 3-week-old mice (p < 0.005). Small increases in Wnt-4 expression (less than 2-fold) occurred during both fetal scarless and postnatal scarring mouse wound repair. The authors' data suggest that TGF-beta directly increases Wnt-4 expression in fetal and postnatal fibroblasts and that Wnt-4 is increased in both fetal and postnatal repair.

α1,3-Galactosyltransferase-Gene Knockout in Cattle using a Single Targeting Vector with loxP Sequences and Cre-Expressing Adenovirus

Gene targeting in large animals has the potential to be useful in medicine as well as in agriculture. Previously, we reported the first successful targeting of the bovine alpha1,3-galactosyltransferase (alpha1,3GT) gene and establishment of a heterozygous knockout cell line. In this report, we generated both heterozygous and homozygous knockout bovine cell lines, and alpha1,3GT-gene knockout cattle. alpha1,3GT gene-disruption was accomplished using primary fetal fibroblasts with a single targeting vector, a promoter-less positive selection vector containing IRES (internal ribosome entry site)-antibiotic-resistance gene (neo) cassette and loxP sequences. At each step in establishing heterozygous and homozygous knockout cell lines, the antibiotic-resistance gene cassette in the targeted allele was removed by a Cre-loxP recombination system that utilizes an adenovirus with transient Cre recombinase expression. A nuclear transfer was performed using alpha1,3GT fetal fibroblasts, and one alpha1,3GT knockout calf was generated but died shortly after birth (day 287). Necropsy revealed normal morphology in all organs. The calf weighed 22.3 kg at birth and this value is within the normal range. The alpha1,3GT knockout- and antibiotic-resistance gene free (alpha1,3GT(-/-)neo-) cells could be cloned normally. Thus, cloned cattle from alpha1,3GT(-/-) neo- cells are potentially safer for human use. Additionally, our strategy is faster and more economical than backcrossing to produce homozygous knockouts. This method should be useful for future production of knockouts of multiple genes in livestock.

94 PRODUCTION OF AN α1,3-GALACTOSYLTRANSFERASE GENE KNOCKOUT CALF USING A SINGLE TARGETING VECTOR AND REJUVENATED CELL LINES

Gene targeting in large animals has the potential to be useful in agriculture. In this study, we examined whether an �1,3-galactosyltransferase-gene knockout (null) calf could be produced using a single targeting vector for disruption of both alleles and a rejuvenation of cell line by production of cloned fetuses. A promoter-less positive selection vector (pGT-22) containing an IRES (internal ribosome entry site)-antibiotic resistance gene(neo) cassette and loxP sequences was used to disrupt the bovine �1,3-galactosyltransferase (�1,3-GT) gene. In gene-targeting (GT), Japanese Black fetal fibroblasts were transfected with pGT-22, and were selected with 0.4 mg mL-1 G418. G418-resistant cells were monitored by PCR and Southern blot analysis. After PCR selection, a portion of the PCR-positive colonies were infected with the adenovirus AxCANCre, which transiently expresses Cre recombinase in the infected cells, to excise the antibiotic-resistance gene cassette (IRES/lacZ-neo) from the targeted allele. The targeted cells in which homologous recombination occurred were used for somatic cell nuclear transfer (SCNT). The cell cycle synchronization of donor cells in the early G1 phase and SCNT were performed according to the established procedure in our laboratory (Urakawa et al. 2004 Theriogenology 62, 714–728). As a result, in the first GT, one PCR-Southern blot-positive clone (0.26%, in 380 G418-resistant colony) was used for SCNT. Of 35 pulsed SCNT embryos, 4 (11.4%) developed to the blastocyst stage and 3 blastocysts were nonsurgically transferred to a recipient heifer; one fetus was recovered on Day 41. The cell line (�1,3-GT+/-) was established and was transfected with pGT-22 in the second GT. As a result, 8 PCR-Southern blot-positive clones (1.6%, in 508 G418-resistant colony) were recovered. The 2nd GT and the following SCNT were accomplished by the same methods used in the 1st GT and SCNT procedure. Of 36 pulsed SCNT embryos, 7 (19.4%) developed to the blastocyst stage and 3 blastocysts were nonsurgically transferred to a recipient heifer; one fetus was recovered on Day 41. The cell line (�1,3-GT-/-) was established, and was used for SCNT to obtain the �1,3-GT homozygous knockout offspring. Finally, 52 of 148 pulsed SCNT embryos (35.1%) developed to the blastocyst stage and 6 cloned embryos were transferred into 3 recipient heifers. A single knockout female offspring with normal morphology in all organs was born (Day 287) but died about one hour after birth. In this study, we established both heterozygous and homozygous �1,3-GT knockout cell lines from primary fetal fibroblasts and produced an �1,3-GT knockout offspring using a single targeting vector and rejuvenated cell lines.

45 NUCLEAR LAMIN A/C EXPRESSION IN BOVINE PARTHENOTES AND NUCLEAR TRANSFER EMBRYOS

Despite the apparent successes of nuclear transfer (NT) technology, numerous recent reports have indicated de-regulation of key gene expression patterns in NT embryos as compared to their in vivo and IVF counterparts. Aberrant expression of lamin A/C has been reported in mouse (Moreira et al. 2003 J. Cell Sci. 116, 3713-3720) and bovine (Sullivan et al. 2004 Biol. Rep. 70, 146-153) NT embryos, leading to the hypothesis that the presence of lamin A/C might affect subsequent development. Lamin A/C expression is a potential marker for reprogramming due to the induced expression and remodeling during differentiation. Previously using immunofluorescence in bovine IVF embryos, we have demonstrated the persistence of lamin A/C until the 2-cell stage (Kelly et al. 2005 Reprod. Fertil. Dev. 17, 205-206). The present study was initiated to further characterize lamin A/C expression in bovine parthenogenetic and NT embryos using a monoclonal antibody specific to lamin A/C. Bovine oocytes were matured in vitro as previously described (Fouladi-Nashta et al. 1998 Biol. Rep. 59, 255-262). NT embryos were constructed using lamin A/C-positive primary bovine fetal fibroblasts and in vitro-matured, enucleated MII bovine oocytes. Oocyte cell couplets were fused at 24 h post onset of maturation 1 h prior to activation. Oocyte activation was achieved with 7% ethanol for 7 min followed by a 6 h incubation in mSOF containing 10 �g/mL cycloheximide and 7.5 �g/mL cytochalasin B for the production of both NT and parthenogenetic embryos. Embryos were cultured in mSOF supplemented with 10% FCS and collected at various stages for immunofluorescence staining. Prior to fixation, embryos were incubated in 2 mg/mL protease to remove the zona pellucida. Samples were fixed in 100% methanol at -20�C for 20 min and then blocked for 1 h (4% goat serum in PBS) at RT. Embryos were then incubated overnight at 4�C with mouse anti-lamin A/C antibody (IgM; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or with blocking solution as a control. Following the primary incubation, embryos were washed extensively in 1% BSA in PBS and then incubated with Cy3 goat anti-mouse IgM (1:400) (Chemicon International, Inc., Temecula, CA, USA) for 1 h at RT. Unbound secondary antibody was removed by washing with 1% BSA in PBS, and embryos were mounted in VectaShield mounting medium containing 42,6-diamidino-1-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Images were viewed using epifluorescence (Leica DMR; Leica Microsystems, Wetzlar, Germany) and confocal microscopy (Leica TCS). Inhibiting protein synthesis during the activation period with cycloheximide had no effect on lamin A/C assembly in 6 h post activation (hpa) parthenogenetic (35/35) and NT (7/7) embryos. The pronuclei of parthenogenetic (30/30) and NT (15/15) zygotes at 22 hpa were also positively labeled for lamin A/C. Nuclear labeling was observed in both parthenogenetic (25/25) and NT (12/12) 2-cell embryos. All parthenogenetic and NT embryos examined from the 4-cell stage through to blastocysts were stained negatively for lamin A/C. These results suggest that lamin A/C present in bovine NT zygotes is not due to aberrant reprogramming and that remodeling of the nuclear lamina occurs correctly in bovine NT embryos.

75 RABBIT NUCLEAR TRANSFER WITH CULTURED SOMATIC CELLS

Cloned rabbits have been obtained by somatic cell nuclear transfer (SCNT) only with fresh, non-cultured cumulus cells (Chesne et al. 2002 Nat. Biotechnol. 20, 366–369). For the purpose of generating transgenic animals by SCNT, donor cells must be cultured and modified prior to use as nuclear donors. The objective of this study was to optimize the SCNT procedure using cultured cumulus or fibroblast cells. MII oocytes were harvested from superovulated Zika rabbits, and maternal chromosomes were removed by demecolcine-assisted enucleation (Yin et al. 2002 Biol. Reprod. 67, 442–446). Two types of somatic cells originating from Ali/Bass rabbits were used as nuclear donors: cumulus cells collected from in vivo-matured oocytes and cultured for 1–5 passages, and primary fetal fibroblasts obtained from Day 16 fetuses and grown to confluence or starved for 4–5 days. Somatic donor cells and recipient cytoplasts were fused with 2 electric pulses (1.95 kV/cm, 25 μs each, 1 s interval). Twenty to 40 min after fusion, cloned embryos were activated first with the same electropulses as for fusion, and then immediately followed by 1 h incubation in 2 mM 6-dimethylaminopurine and 5 μg/mL cytochalasin B in culture medium (B2 medium supplemented with 10% FCS). Cloned embryos were either transferred at the 2- and 4-cell stage to asynchronized recipients or cultured in vitro for 6 days. Data were compared using chi-square test, and differences were considered significant when P &lt; 0.05. Our results demonstrate that cloned rabbits can be produced by SCNT with cultured cells but the efficiency of this technique is still very low irrespective of the type of donor cells. Table 1. Development of cloned embryos derived from somatic cells This research was supported by the Therapeutic Human Polyclonals, Inc.

Cigarette Smoke in Research

In this issue of the AJRCMB, Kim and colleagues (see article beginning on page 483) demonstrate that cigarette smoke extract (CSE) can cause reversible DNA damage to primary human fetal fibroblasts. The use of crude cigarette smoke extract on cells in culture is, well, crude, and has often been criticized. In this particular case, the novel, mechanistic information with potential relevance to lung pathobiology merited publication. Nevertheless, we asked the senior author to discuss and justify the use of CSE as a model system.

The Search for Biological Truth Using Imperfect Models in an Imperfect World

In this issue of the AJRCMB, Kim and colleagues (see article beginning on page 483) demonstrate that cigarette smoke extract (CSE) can cause reversible DNA damage to primary human fetal fibroblasts. The use of crude cigarette smoke extract on cells in culture is, well, crude, and has often been criticized. In this particular case, the novel, mechanistic information with potential relevance to lung pathobiology merited publication. Nevertheless, we asked the senior author to discuss and justify the use of CSE as a model system.

Expression profiles of p53 and p66 shc during oxidative stress-induced senescence in fetal bovine fibroblasts

Somatic cells undergo a permanent cell cycle arrest, called cellular senescence, after a limited number of cell divisions in vitro. Both the tumor suppressor protein p53 and the stress-response protein p66 shc are suggested to regulate the molecular events associated with senescence. This study was undertaken to investigate the effect of different oxygen tensions and oxidative stress on cell longevity and to establish the role of p53 and p66 shc in cells undergoing senescence. As a model of cellular senescence, primary fetal bovine fibroblasts were cultured in either 20% O 2 or 5% O 2 atmospheres until senescence was reached. Fibroblasts cultured under 20% O 2 tension underwent senescence after 30 population doublings (PD), whereas fibroblasts cultured under 5% O 2 tension did not exhibit signs of senescence. Oxidative stress, as measured by protein carbonyl content, was significantly elevated in senescent cells compared to their younger counterparts and to fibroblasts cultured under 5% O 2 at the same PD. p53 mRNA gradually decreased in 20% O 2 cultured fibroblasts until senescence was reached, whereas p53 protein levels were significantly increased as well as p53 phosphorylation on serine 20, suggesting that p53 might be stabilized by posttranslational modifications during senescence. Senescence was also associated with high levels of p66 shc mRNA and protein levels, while the levels remained low and stable in dividing fibroblasts under 5% O 2 atmosphere. Taken together, our results show an effect of oxidative stress on the replicative life span of fetal bovine fibroblasts as well as an involvement of p53, serine 20-p53 phosphorylation and p66 shc in senescence.

Targeted Correction of Single-Base-Pair Mutations with Adeno-Associated Virus Vectors under Nonselective Conditions

Recombinant adeno-associated virus (rAAV) vectors possess the unique ability to introduce genetic alterations at sites of homology in genomic DNA through a mechanism thought to predominantly involve homologous recombination. We have investigated the efficiency of this approach using a mutant enhanced green fluorescent protein (eGFP) fluorescence recovery assay that facilitates detection of gene correction events in living cells under nonselective conditions. Our data demonstrate that rAAV infection can correct a mutant eGFP transgene at an efficiency of 0.1% in 293 cells, as determined by fluorescence-activated cell-sorting analysis. Gene repair was also confirmed using clonal expansion of GFP-positive cells and sequencing of the eGFP transgene. These results support previous findings demonstrating the efficacy of rAAV for gene targeting. In an effort to improve gene-targeting efficiencies, we evaluated several agents known to increase rAAV transduction (i.e., expression of an expressed gene), including genotoxic stress and proteasome inhibitors, but observed no correlation between the level of gene repair and rAAV transduction. Interestingly, however, our results demonstrated that enrichment of G(1)/S-phase cells in the target population through the addition of thymidine moderately (approximately 2-fold) increased gene correction compared to cells in other cell cycle phases, including G(0)/G1, G(1), and G(2)/M. These results suggest that the S phase of the cell cycle may more efficiently facilitate gene repair by rAAV. Transgenic mice expressing the mutant GFP were used to evaluate rAAV targeting efficiencies in primary fetal fibroblast and tibialis muscles. However, targeting efficiencies in primary mouse fetal fibroblasts were significantly lower (approximately 0.006%) than in 293 cells, and no correction was seen in tibialis muscles following rAAV infection. To evaluate the molecular structures of rAAV genomes that might be responsible for gene repair, single-cell injection studies were performed with purified viral DNA in a mutant eGFP target cell line. However, the failure of direct cytoplasm- or nucleus-injected rAAV DNA to facilitate gene repair suggests that some aspect of intracellular viral processing may be required to prime recombinant viral genomes for gene repair events.

Transient alteration of cell fate using a nuclear and cytoplasmic extract of an insulinoma cell line

We report a transient modulation of cell fate in fibroblasts briefly exposed to an extract derived from the rat insulin-producing β cell line, INS-1E. Primary fetal rat fibroblasts were reversibly permeabilized with Streptolysin O, incubated for 1 h in a 15,000 g INS-1E nuclear and cytoplasmic extract, resealed, and cultured. A first marker of change in cell fate was a reduction of cell and nuclear size within days of exposure to extract such that in some instances the fibroblasts resembled INS-1E cells. Second, two β cell transcripts, Pdx-1 and insulin, were detected in the fibroblasts for up to 4 weeks. Third, (pro)insulin labeling was detected in 5–30% of the cells for a period of 8–14 days after incubation in extract. These phenotypes were absent from fibroblasts exposed to heat-treated INS-1E extracts, a human fibroblast cell line-derived extract or buffer. The results indicate that the extract of an insulinoma-derived cell line can promote at least a transient modification of cell fate towards a β cell phenotype in non-β cells. Because they are easily accessible, cell extracts may represent a practical source of material for investigating the mechanisms of alteration of a nuclear and cellular program.

Nuclear localization signal and cell synchrony enhance gene targeting efficiency in primary fetal fibroblasts.

The use of primary somatic cells in nuclear transfer procedure has opened a new opportunity to manipulate domestic animal genomes via homologous recombination. To date, while a few loci have been targeted in somatic cells using similar enrichment strategies as those used in mouse ES cells, there have been problems of low efficiency, mixed targeted and non-targeted cells within a colony and difficulties in cloning the cell after targeting. Utilizing the hypoxanthine guanine phosphoribosyl transferase (HPRT) as a test locus, it was determined that while no targeted colonies were identified using a conventional targeting construct, an average of 1 per million targeted cells were identified when a nuclear localization signal (nls) was added to the construct. When the nls was combined with cell synchronization using a thymidine block, targeting efficiency increased 7-fold. Moreover, the number of random integrants decreased by over 54-fold resulting in a 1:3 targeted to random integration ratio. This method should facilitate the application of homologous recombination to primary somatic cells.

80DEVELOPMENTAL POTENTIAL OF BOVINE NUCLEAR TRANSFER EMBRYOS DERIVED FROM FETAL FIBROBLASTS TRANSDUCED WITH SIMIAN VIRUS 40 LARGE T ANTIGEN

Primary cultured cells with limited lifespan are generally used as donors for nuclear transfer (NT). Immortalization or even extension of their proliferative capacity would provide an additional time-window for various genetic manipulations prior to NT. Previously, we reported that a spontaneously immortalized mammary epithelial cell line failed to support development of reconstructed embryos into blastocysts [Zakhartchenko et al., 1999 Mol. Reprod. Dev. 54, 264–272]. In contrast, telomerase-immortalized sheep fibroblasts could be substantially reprogrammed, although they were not fully competent for NT since no fetuses survived beyond 40 days of development [Cui et al., 2003 Biol. Reprod. 69, 15–21]. Simian virus 40 large T antigen (SV40Tag), a viral oncoprotein, is known to immortalize human diploid fibroblasts by soaking up the cellular negative growth regulators, pRb, p53, and some related factors to enable cells to grow continuously [Kim et al., 2001 Exp. Mol. Med. 33, 293–298]. In this study we examined the developmental competence of bovine NT embryos derived from SV40Tag-transduced fetal fibroblasts. Primary fetal fibroblasts (BFF) obtained from a 49-day-old fetus were first transduced with the green fluorescent protein (GFP) gene and then with SV40Tag using a replication-defective retrovirus. GFP-SV40Tag-positive cells (BFF-GFP-Tag), which can proliferate over 50 passages, were cultured until confluence and then used for nuclear transfer at passages 27–30. As control, confluent GFP-positive cells (BFF-GFP) and BFF, which stopped proliferation after 28 passages, were used at passages 4–6 and 1–2, respectively. Nuclear transfer and embryo culture procedures were essentially as described previously [Shi et al., 2003 Biol. Reprod. 69, 301–309]. Data were compared using chi-square test; differences were considered significant for P&amp;lt;0.05. There were no significant differences in the fusion and cleavage rates between all three groups [BFF-GFP-Tag: 233/248 (94%) and 167/233 (72%); BFF-GFP: 274/294 (93%) and 216/274 (79%); BFF: 129/136 (96%) and 97/129 (75%)]. However, development to the blastocyst stage was significantly lower (P&amp;lt;0.05) in the BFF-GFP-Tag group [5/233 (2%)] than in the BFF-GFP and BFF groups [110/275 (40%) and 53/129 (41%), respectively)]. From our results we conclude that SV40Tag-transduced bovine fetal fibroblasts with high proliferative potential may have lost the ability to respond to the normal cell cycle controls and, as a consequence, have low developmental potential for nuclear transfer.

343NUCLEAR LOCALIZATION SIGNAL AND CELL SYNCHRONY ENHANCES GENE TARGETING EFFICIENCY IN FETAL BOVINE FIBROBLASTS

The use of primary somatic cells for nuclear transfer has facilitated the manipulation of the domestic animal genome via homologous recombination. Yet, the absolute frequency of homologous recombination (HR) in somatic cells is two orders of magnitude lower than in ES cells whereas frequencies of non-homologous end joining are higher. While a few loci have been targeted in somatic cells using enrichment strategies similar to those used in mouse ES cells, there have been problems of low efficiency, mixed targeted and non-targeted cells, and difficulties in cloning the cell after targeting. We present evidence that the use of a nuclear localization signal (nls) and S-phase cell cycle synchronization by thymidine block enhances targeting efficiency at the hypoxanthine phosphoribosyl transferase locus in primary fetal bovine fibroblasts. We designed two hypoxanthine phosphoribosyl transferase (HPRT)-targeting constructs, HPRT-DEx6 and HPRT-DEx6-nls. Both constructs have a 31-bp deletion and a PGK-puro insertion in exon 6 to ensure inactivation of the HPRT locus. Additionally, the HPRT-DEx6-nls construct contains a 180-bp cassette comprised of two 72-bp tandem repeats from SV40 enhancer known to act as nuclear localization signal. Diploid male cells that undergo targeted gene disruption at the single copy X-linked HPRT locus can be selected with 8-Azaguanine (8-AG) as HPRT cells are resistant to 8-AG; all transformants, random and targeted, can be selected in puromycin. Male primary bovine fibroblasts were electroporated with linearized targeting constructs, and plated in media containing puromycin or puromycin plus 8AG. All experiments were done in triplicate and data were analyzed by two-way ANOVA with NLS and cell synchrony as the two factors. Significance was set at P&amp;lt;0.01. While the total number of insertions (random plus targeted) with both constructs were equivalent, the HPRT-DEx6-nls construct produced a significantly higher number of targeted colonies (1–2 8AG-resistant colonies per 9.5×106 cells) than HPRT-DEx6 where no targeted events were seen. Cells were synchronized in the S-phase of the cell cycle by a 2mM thymidine treatment for 24 hours and electroporated with the linearized targeting constructs. Compared to non-synchronous cells, the total number of insertions (random plus targeted) was reduced by 59-fold in constructs with or without nls (P&amp;lt;0.01), while targeted insertions increased 6-fold in the HPRT-DEx6-nls construct, from an average of 10 per million cells without nls to 7.6 per 10 million with nls (P&amp;lt;0.01). All 8AG-resistant colonies were verified by Long Range-PCR, and PCR products confirmed by end sequencing. This finding has important implications for targeting in somatic cells, as with a drastic reduction in the number of random insertions, and increased targeting due to the presence of the nls, identification of a targeted colony is greatly facilitated even in cases where no enrichment protocols are available.

UP1 extends life of primary porcine fetal fibroblasts in culture.

Genetic modification of somatic cell nuclei and subsequent nuclear transfer has opened an opportunity to create gene-targeted animals. However, somatic cells have a limited life span in culture and it is not possible to introduce precise genetic changes in both alleles in this narrow time window. To increase the life span of somatic cell in culture, both genetic and chemical approaches have been tried with varying success. Here, we report the effect of two anti-oxidants, glutathione and n-t-butyl hydroxylamine, and of the expression of UP1, a shortened derivative of heterogeneous nuclear riboprotein (hnRNP)A1, on the life extension of primary porcine fibroblasts in culture. Under our experimental conditions, the use of anti-oxidants did not result in any prolongation of the life span. In contrast, UP1 expression increased the life span significantly. While most control cells stopped growing by PDL 20, and none survived beyond PDL 35, 100% of UP1-expressing clones reached PDL50, and 40% made it to PDL65. The five UP1-expressing clones were karyotyped at PDL 50. While all of them had a range of numerical chromosomal abnormalities, two clones retained 30-40% normal cells, all the cells in other three clones had abnormal chromosome numbers. Thus, expression of UP1 may be useful in extending the life span of somatic cells in culture. This, in turn, will facilitate the process of gene targeting in this cell type.

Prostaglandin E2 inhibits fibroblast to myofibroblast transition via E. prostanoid receptor 2 signaling and cyclic adenosine monophosphate elevation.

Myofibroblasts, the hallmark of fibrotic disease, contribute to the pathology of fibrosis by secreting large amounts of extracellular matrix and contributing to alveolar contraction. Myofibroblasts are characterized by the expression of alpha-smooth muscle actin (alpha-SMA), a contractile protein normally associated with smooth muscle cells. Transforming growth factor-beta1 (TGF-beta1) is a well characterized profibrotic cytokine that induces myofibroblast transformation both in vitro and in vivo. We report here that the lipid mediator prostaglandin E2 (PGE2) inhibits TGF-beta1-induced expression of alpha-SMA in primary fetal and adult lung fibroblasts. This inhibition of alpha-SMA expression is associated with a reduction in the expression of collagen I. Inhibitory actions of PGE2 are mediated via E prostanoid receptor 2 (EP2) signaling, but not by EP3 signaling, and increases in cyclic adenosine monophosphate production. The inhibitory effects of PGE2 on TGF-beta1-induced alpha-SMA expression are mimicked by an EP2 selective agonist, butaprost, and by forskolin-induced direct activation of adenyl cyclase. An EP2 antagonist blocks the inhibitory effects of PGE2, and an EP3 agonist does not inhibit TGF-beta1-mediated increases in alpha-SMA expression. Our results demonstrate that PGE2 inhibits transition of fibroblasts to myofibroblasts by an EP2 receptor-activated pathway. Augmenting this pathway may serve as a potent antifibrotic therapeutic strategy.

Induction of a senescent-like phenotype does not confer the ability of bovine immortal cells to support the development of nuclear transfer embryos.

Previously, we reported that cloned embryos derived from an immortalized bovine mammary epithelial cell line (MECL) failed to develop beyond 12- to 16-cell stage. To analyze whether induction of a senescent-like phenotype in MECL can improve their ability to support the development after transfer into enucleated oocytes, we treated MECL with DNA methylation inhibitor 5-aza-2-deoxycytidine (Aza-C), histone deacetylase inhibitors trichostatin A (TSA), sodium butyrate (NaBu), or 5-bromodeoxyuridine and used those cells for nuclear transfer. Primary bovine fetal fibroblasts (BFF) were used as control. All agents were capable to induce features of senescence including reduced cell proliferation, enlarged cell size with a considerable proportion of cells stained positive for acidic senescence-associated beta-galactosidase and G1/S cell cycle boundary arrest in MECL. Aza-C treatment induced genome demethylation. Acetylation of H3 and H4 was increased after TSA treatment in both MECL and BFF, whereas no obvious changes in global H3 or H4 acetylation were detected after NaBu treatment. Nuclear transfer experiments following diverse treatments demonstrated that the induced senescent-like phenotype of MECL did not confer their ability to support embryonic development, although 7.3% of reconstructed embryos derived from NaBu-treated cells developed to morula stage. Intriguingly, a much higher proportion of cloned embryos developed to blastocysts when using NaBu-treated BFF, compared with using untreated BFF (59% versus 26%). Our results suggest that the developmental failure of donor nuclei from bovine immortal cells could not be reversed by induction of senescent-like phenotype. The beneficial effect of NaBu on the developmental potential of cloned embryos reconstructed from BFF merits further studies.