Toward an ideal animal model to trace donor cell fates after stem cell therapy: production of stably labeled multipotent mesenchymal stem cells from bone marrow of transgenic pigs harboring enhanced green fluorescence protein gene.

The discovery of postnatal mesenchymal stem cells (MSC) with their general multipotentiality has fueled much interest in the development of cell-based therapies. Proper identification of transplanted MSC is crucial for evaluating donor cell distribution, differentiation, and migration. Lack of an efficient marker of transplanted MSC has precluded our understanding of MSC-related regenerative studies, especially in large animal models such as pigs. In the present study, we produced transgenic pigs harboring an enhanced green fluorescent protein (EGFP) gene. The pigs provide a reliable and reproducible source for obtaining stable EGFP-labeled MSC, which is very useful for donor cell tracking after transplantation. The undifferentiated EGFP-tagged MSC expressed a greater quantity of EGFP while maintaining MSC multipotentiality. These cells exhibited homogeneous surface epitopes and possessed classic trilineage differentiation potential into osteogenic, adipogenic, and chondrogenic lineages, with robust EGFP expression maintained in all differentiated progeny. Injection of donor MSC can dramatically increase the thickness of infarcted myocardium and improve cardiac function in mice. Moreover, the MSC, with their strong EGFP expression, can be easily distinguished from the background autofluorescence in myocardial infarcts. We demonstrated an efficient, effective, and easy way to identify MSC after long-term culture and transplantation. With the transgenic model, we were able to obtain stem or progenitor cells in earlier passages compared with the transfection of traceable markers into established MSC. Because the integration site of the transgene was the same for all cells, we lessened the potential for positional effects and the heterogeneity of the stem cells. The EGFP-transgenic pigs may serve as useful biomedical and agricultural models of somatic stem cell biology.

Porcine peroxisome proliferator-activated receptor delta mediates the lipolytic effects of dietary fish oil to reduce body fat deposition.

Peroxisome proliferator-activated receptor delta promotes fatty acid catabolism and energy expenditure in skeletal muscle and adipose tissues. A ligand for PPARdelta is required to activate PPARdelta function. Polyunsaturated fatty acids are potential ligands for PPARdelta activation. The current experiment was designed to determine the potential for PUFA, particularly from dietary fish oil, to activate porcine PPARdelta in vivo. Transgenic mice were generated to overexpress porcine PPARdelta in the adipose tissue. Mice were fed a high-saturated fat (13% beef tallow), or high-unsaturated fat (13% fish oil) diet, or a diet containing 4 mg/kg of a PPARdelta ligand (L165041) for 4 mo. Compared with beef tallow feeding, fish oil feeding reduced fat mass and decreased (P < 0.05) plasma triacylglycerol and FFA concentrations in the transgenic mice. Adipose tissue expression of genes involved in adipogenesis (i.e., lipoprotein lipase and adipocyte fatty acid-binding protein) was decreased in transgenic mice fed fish oil or the PPARdelta ligand. In the same mice, expression of the lipolytic gene, hormone-sensitive lipase was increased (P < 0.05). Fish oil feeding also stimulated expression of genes participating in fatty acid oxidation in the liver of transgenic mice compared with wild-type mice. Overall, these results indicate that PUFA may serve as natural and effective regulators of lipid catabolism in vivo and many of these effects may be generated from activation of PPARdelta.

The effect of feed restriction on expression of hepatic lipogenic genes in broiler chickens and the function of SREBP1.

To study the role of sterol regulatory element-binding proteins (SREBP) in lipogenesis and cholesterol synthesis in the chicken, two experiments were carried out. In the first study, seven-week-old broilers (n=16) were allocated into 2 groups, fasted for 24 h or refed for 5 h after a 24 h fasting. The mRNA concentrations for SREBPs and other lipogenic genes in the liver were determined by quantitative real time PCR. The hepatic mRNA relative abundance of lipogenic genes and genes involved in cholesterol synthesis were significantly greater (p<0.001) in the refed broilers. Similar results were demonstrated with Northern analysis. The data suggest that in the liver of fasted broilers, genes associated with lipogenesis and cholesterol biosynthesis were inhibited. Indeed, the mRNA concentrations for fatty acid synthase (FAS), malic enzyme, and stearoyl coenzyme A desaturase were almost undetectable after the 24 h fasting. The data also demonstrated that the expression of lipogenic genes coordinate well as a group during the refeeding period. Second, three small interfering RNA (siRNA) oligonucleotides against SREBP1 were designed to be used in transfecting a chicken hepatocarcinoma cell line LMH. One of the three siRNAs effectively reduced SREBP1 mRNA concentration (p<0.01). The acetyl coenzyme A carboxylase(alpha) (ACC(alpha)) mRNA was also significantly reduced by the SREBP1 siRNA treatment, suggesting that SREBP1 can upregulate the expression of this lipogenic gene. This siRNA, however, did not affect the mRNA for FAS. Taken together, the RNA interference study showed that SREBP1 has the ability to regulate the expression of ACC(alpha). This study has helped us understand more about the function of SREBP1 and the physiology of the broiler chickens.

Expressed transcripts associated with high rates of egg production in chicken ovarian follicles.

The purpose of this study was to characterize differentially expressed transcripts associated with varying rates of egg production in Taiwan country chickens. Ovarian follicles were isolated from two strains of chicken which showed low (B) or high (L2) rates of egg production, then processed for RNA extraction and cDNA library construction. Three thousand and eight forty clones were randomly selected from the cDNA library and amplified by PCR, then used in microarray analysis. Differentially expressed transcripts (P<0.05, log(2)> or = 1.75) were sequenced, and aligned using GenBank. This analysis revealed 20 non-redundant sequences which corresponded to known transcripts. Eight transcripts were expressed at a higher level in ovarian tissue prepared from chicken strain B, and 12 transcripts were expressed at a higher level in L2 birds. These differential patterns of expression were confirmed by semi-quantitative RT-PCR. We show that transcripts of cyclin B2 (cycB2), ferritin heavy polypeptide 1 (FTH1), Gag-Pol polyprotein, thymosin beta4 (TB4) and elongation factor 1 alpha1 (EEF1A1) were enriched in B strain ovarian follicles. In contrast, thioredoxin (TXN), acetyl-CoA dehydrogenase long chain (ACADL), inhibitor of growth family member 4 (ING4) and annexin II (ANXA2) were expressed in at higher levels in the L2 strain. We suggest that our approach may lead to the isolation of effective molecular markers that can be used in selection programs in Taiwan country chickens.

The differential expression of hepatic genes between prelaying and laying geese.

Suppression subtractive hybridization was used to detect differential expression of genes in the livers of laying and prelaying geese. Liver tissues from prelaying and laying geese were dissected for mRNA extraction. The cDNA, reverse transcribed from liver mRNA of prelaying geese, was subtracted from the cDNA generated from the laying geese (forward subtraction). Five hundred seventy-six clones with possible differentially expressed gene fragments were observed by forward subtraction hybridization. After differential screening using the reverse and forward subtraction cDNA, 164 clones were subjected to gene sequence determination and further analysis. Using Northern analysis, 5 known and 8 unknown genes were shown to be highly expressed in the livers of laying geese compared with prelaying geese. Vitellogenin I, apoVLDL-II, ethanolamine kinase, G-protein gamma-5 subunit, and leucyl-tRNA synthase were highly expressed in the livers of laying geese compared with that from the prelaying geese (P<0.05). The expression of these known genes suggests that their function in the liver of laying geese is primarily involved in lipid and lipoprotein metabolism. Several of these differentially expressed genes were found to be responsive to estrogen stimulation, confirming the involvement of these genes in the egg-laying function of the goose.

The development of an immunoblotting assay for the quantification of liver fatty acid-binding protein during embryonic and early posthatch development of turkeys (Meleagridis gallopavo).

Turkey (Meleagridis gallopavo) liver cytosolic fatty acid binding protein (FABP) was purified and used as a standard for quantification. An immunoblotting procedure was developed to study the ontogeny of liver cytosolic FABP during embryonic and early posthatch development in turkey poults. Liver FABP activity was also determined indirectly through the use of gel filtration chromatography followed by a ligand-binding assay. The specific activity of liver FABP (ng/mg of cytosolic protein) increased with length of incubation, peaking initially at Day 22, declining between Days 22 and 25, and increasing again from hatch (Day 28) to 6 d posthatch. The specific activity of liver FABP increased 12-fold between Day 13 of incubation and 6 d posthatch compared with total activity, which increased from 946 to 1.01 x 10(6) ng/liver during the same period, a 1,067-fold increase. The results from both analytical procedures were similar, suggesting that the immunoblot method could be used to quantify liver FABP concentrations. The observed increases in FABP activity throughout the embryonic period and first days after hatching paralleled increases in liver lipid concentration. Therefore, liver FABP may be associated with hepatocyte fatty acid transport and metabolism during the latter stages of incubation and early posthatch period.

The ontogeny of fatty acid-binding protein in turkey (Meleagridis gallopavo) intestine and yolk sac membrane during embryonic and early posthatch development.

Experiments were conducted to confirm the existence and ontogeny of fatty acid binding protein (FABP) in the intestine and yolk sac membrane of turkey poults (Meleagridis gallopavo) during embryonic and early posthatch development. Intestinal (I-) FABP was measured using an immunoblot procedure incorporating anti-chick liver (L-)FABP antisera. FABP activity in both tissues was also confirmed with a ligand-binding assay incorporating 14C-oleic acid. I-FABP did not cross-react with chick L-FABP antisera until hatch, embryonic day 28 (ED 28), after which there was a 39% increase in I-FABP concentration through the first 3 d posthatch (PD 3). FABP concentration calculated on a total intestinal basis (ng/intestine), however, increased 10-fold through PD 6. Specific activity [disintegrations per minute (dpm)/ mg cytosolic protein] was greatest at hatch and decreased slightly thereafter, whereas specific activity of FABP in the yolk sac membrane peaked between ED 16 and ED 19 and then declined. Total yolk sac activity (dpm/yolk sac membrane), however, plateaued at ED 22 before declining to low levels by PD 3, coincident with the period of maximal lipid transfer out of the yolk.

UV inducibility of rat proliferating cell nuclear antigen gene promoter.

Proliferating cell nuclear antigen (PCNA), also known as a cofactor of DNA polymerase delta, is required for eukaryotic cell DNA synthesis and nucleotide excision repair. Expression of PCNA gene is growth-regulated and UV inducible. In our previous study, we have observed that the rat PCNA promoter has the serum responsiveness. In this study, we demonstrate its UV inducibility in CHO.K1 cells. The UV induction of the rat PCNA promoter activity was dose-dependent in the cells synchronized at different phases. In addition, the sequences of the promoter responsible for the UV inducibility were delimited to the region between nucleotides -70 and +125, which contains an AP-1 site and a downstream proximal ATF/CRE site. While mutation of the AP-1 site abrogated the UV inducibility, mutation of the ATF/CRE site enhanced the UV inducibility, suggesting that the two sites play different roles in the UV induction of the promoter. In addition, the role of p53 in the UV induction of rat PCNA promoter was investigated. We found that exogenous p53 was unable to mimic the UV irradiation to induce rat PCNA promoter and that the UV induction of the rat PCNA promoter was seen in p53 deficient cells. Therefore, it is unlikely that the UV induction of the rat PCNA promoter is p53 dependent.

Inclusion of coconut oil in diets for turkey breeders and its effects on embryonic yolk and liver fatty acids.

Turkey hens were fed either a standard breeder diet (CON, myristic acid, C14.0, 1.1%; palmitic acid, C16:0, 16.8%; oleic acid, C18:1, 23%; linoleic acid, C18:2, 48.7%) or a diet containing 5% coconut oil (COCO) enriched with medium chain fatty acids (MCFA; lauric acid, C12:0, 22.6%; C14:0, 10.8%; C16:0, 12.5%; C18:1, 14.8%; C18:2, 24.6%). After 10 d on the diets, fresh eggs were collected for yolk lipid and fatty acid (FA) determination. An additional 60 to 95 eggs were incubated and the FA profiles of the neutral lipid (NL) and phospholipid (PL) fractions of yolk sac and liver lipids were determined. The NL fraction of the yolk sac from CON eggs contained less C12:0 (0 vs 0.49%) and C14:0 (0.7 vs 4.6%) and more C18:1 (41.3 vs 37.5%). The PL fraction of the yolk sac from both treatments contained < 1% C14:0, and there was less than a 2% difference between treatments in other FA concentrations. The hepatic NL fraction from both treatments contained < 1% C14:0 and only C18:1 showed > 1% differences between treatments (Control = 59.9%; COCO = 56.62%). There were no dietary effects on the FA profile of hepatic PL. The presence of only minimal quantities of MCFA in hepatic NL and PL suggests that absorbed yolk sac MCFA are extensively metabolized during embryonic development.

Serum responsiveness of the rat PCNA promoter.

Expression of proliferating cell nuclear antigen (PCNA) gene is growth-regulated. The growth dependence of the rat PCNA gene promoter activity was investigated. Cultured cells were transfected with the promoter containing plasmid and recovered for 48 h in serum-free medium to become quiescent. Cells were then cultured in serum-containing medium and harvested at certain intervals after serum-stimulation, and the promoter-directed chloramphenicol acetyltransferase activities in cell extracts were measured. The promoter used in this study contained sequences between -693 and +125 in relation to the transcription initiation site. The promoter activity was found to be serum-responsive. However, the serum-responsiveness of the promoter became less obvious when the amount of the promoter increased; meanwhile, the promoter became active in the control unstimulated (or quiescent) cells. It was suspected that the dosage effect was due to the titration of the negative regulatory factor in quiescent cells. The titration experiment with a reporterless construct as competitor for regulatory factors showed that the excess of promoter molecules reduced the promoter activity in serum-stimulated cells, while causing a transiently increase of promoter activity in quiescent cells. Based on these results, it is postulated that the serum-responsiveness of the rat PCNA promoter is controlled by both negative and positive regulatory factors. Consistent with this proposition, promoter binding proteins of 105 and 114 kDa were identified only in serum-stimulated and quiescent cells, respectively, in addition to several other promoter binding proteins (ranging from 76 to 110 kDa) which were seen in both serum-stimulated and quiescent cells.