Β-galactosidase Positive Cells Research Articles

Bone Marrow Osteoclasts Stain Positive with X-Gal: Implications for the Tracking of LacZ-Labelled Cells.

The E.coli gene Lac-Z encodes for β-galactosidase and is often used to track transgenic cells after transplantation in vivo or to analyze temporospatial gene expression patterns by coupling expression of specific target genes to β-galactosidase activity. Several groups have reported endogenous β-galactosidase activity as a confounding factor in the study of different soft tissues. However, the typical background to be found on bone marrow sections when using the chromogenic substrate 5-Bromo-4-chloro-3-indolyl β-D-Galactoside (X-Gal) has not been described before. We demonstrate here that X-Gal stained wild type murine bones display a typical epiphyseal β-gal positive stain line that can already be seen on whole mount preparations. With light microscopy, the β-gal positive cells are located along the endosteum adjacent to trabecular bone and in particularly high concentrations in the epiphyseal resorption zone. In addition, the β-gal positive cells bear the typical features of osteoclasts in that they are multinucleated and display the typical foamy cytoplasm of osteoclasts. When an antibody against a well-known and previously published marker of the vasculature, pan-endothelial cell antigen (clone MECA32), was used in wild type bone, the X-Gal positive osteoclasts could be found in their typical location in epiphyseal resorption pits and closely located to the bone marrow vasculature. This provides further evidence that the X-Gal positive cells are indeed osteoclasts. Finally, double staining for tartrate resistant acid phosphatase (TRAP) and with X-Gal in wild type murine bones resulted in a complete match of the staining pattern and provided formal proof of the hypothesis that osteoclasts have strong β-galactosidase activity. In order to establish common knowledge of this potentially misleading artifact, we systematically examined the bones of different wild type mouse strains at various stages of development starting at the neonatal period. The same pattern of background staining was observed in C57bl/6, FvB/NJ, 129P3/J, and CD1 strains of mice. In addition, we examined the effect of pH and time of exposure to substrate and observed no changes in endogenous mammalian b-gal activity.In conclusion, we show that osteoclasts in bone marrow sections specifically and robustly stain blue with X-Gal. This leads to a typical background when bone marrow is examined that is present from the first day post partum throughout the adult life of experimental mice and can easily be confused with transgenic, bacterial β-galactosidase expressing hematopoietic or stromal cells. We conclude from these data that there is a field-wide need for strenuous controls and further classification when evaluating β-galactosidase positive bone marrow cells. Verifiable bacterial β-galactosidase positive bone marrow cells should be further identified using immunohistological or other approaches. Alternatively, β-galactosidase positive cells should be proven not to be osteoclasts by co-staining or by staining of adjacent sections.

In vivo transgene expression from an adenoviral vector is altered following a 6-OHDA lesion of the dopamine system

We have investigated the in vivo dynamics of an adenovirus-based, LacZ expressing vector, RAd36, at different doses, when injected unilaterally into the corpus striatum of normal rats. We have further investigated the characteristics of this vector in the presence of a 6-OHDA lesion of the nigrostriatal pathway. The dopamine-depleting lesion had an effect on both the number and the distribution of cells transduced by the adenoviral vector. The lesioned side of the brain contained significantly greater numbers of β-galactosidase positive cells than the unlesioned side at 3 days, 1 week and 4 weeks post-injection and the distribution of transduced cells was altered by the presence of a dopamine lesion. We conclude that the increased levels of transgene expression seen in the lesioned hemisphere are due to a change in the diffusion characteristics of the injected vector in the lesioned hemisphere. These results indicate that, when investigating the use of virus-based vectors, ultimately for use in gene therapies in the CNS, the in vivo dynamics of the vector need to be assessed not only in the normal brain, but also in the pathological brain state such as animal models of target diseases.

Long term phenobarbital administration does not promote the multiplication of hepatocytes replicating after single cyproterone acetate administration

Cyproterone acetate (CPA) is a synthetic antiandrogenic compound which is widely used in clinic but suspected to be hepatocarcinogenic. CPA is also mitogenic in rat liver. Using genetic labeling of dividing cells, we examined whether hepatocytes dividing in response to acute CPA administration could give rise to preneoplastic foci after administration of a tumor promoter: phenobarbital. CPA was administered orally to rats and dividing hepatocytes were genetically labeled using retroviral vectors carrying the β-galactosidase gene. After labeling rats were given phenobarbital for 10 months and sacrificed. The presence of β-galactosidase labeled hepatocytes as well as preneoplastic hepatocytes was assessed by immunohistochemistry. Genetic labeling of hepatocytes was obtained in all animals. At the end of phenobarbital administration, no hepatic tumors were observed. Preneoplastic foci were not increased in treated animals as compared to control rats. Moreover β-galactosidase positive hepatocytes were never detected in preneoplastic foci. Finally, the size of the β-galactosidase postive clusters was smaller in treated animals as compared to control rats. We conclude that acute CPA administration is not carcinogenic in rat liver and does not initiate preneoplastic hepatocytes capable to give rise to foci after phenobarbital promotion. Therefore the mitogenic property of CPA is distinct from its putative carcinogenic activity. Finally, analysis of the size of β-galactosidase positive cells clusters demonstrate that phenobarbital does not induce hepatocyte proliferation in rats.

Cellular responses in experimental liver injury

Background/Aims Mature hepatocytes divide to restore liver mass after injury. However, when hepatocyte division is impaired by retrorsine poisoning, regeneration proceeds from another cell type: the small hepatocyte-like progenitor cells (SHPCs). Our aim was to test whether SHPCs could originate from mature hepatocytes. Methods Mature hepatocytes were genetically labeled using retroviral vectors harboring the β-galactosidase gene. After labeling, retrorsine was administered to rats followed by partial hepatectomy to trigger regeneration. A liver biopsy was performed one month after surgery and rats were sacrificed one month later. Results We observed the proliferation of small hepatocytes arranged in clusters in liver biopsies. These cells expressed Ki67 antigen and displayed high mitotic index. At sacrifice, regeneration was completed and clusters had merged. A significant proportion of clusters also expressed β-galactosidase demonstrating their origin from labeled mature hepatocytes. Finally, the overall proportion of β-galactosidase positive cells was identical at the time of hepatectomy as well as in liver biopsy and at sacrifice. Conclusions The constant proportion of β-galactosidase positive cells during the regeneration process demonstrates that mature hepatocytes are randomly recruited to proliferate and compensate parenchyma loss in this model. Furthermore, mature hepatocytes are the source of SHPC after retrorsine injury.

Poly(methylidene malonate 2.1.2) nanoparticles: a biocompatible polymer that enhances peri-adventitial adenoviral gene delivery

Vascular gene therapy is currently limited by low and transient levels of gene transfection. The objectives of this study were to determine whether peri-adventitial delivery of adenovirus coupled to nanoparticles could improve transfection efficiency and duration. Adenovirus was absorbed to the surface of nanoparticles that were made from poly(methylidene malonate)2.1.2 (PMM2.1.2). These complexes were found to have good adhesive properties to both cultured vascular smooth muscle cells and to the luminal and adventitial layers of excised rabbit carotid arteries. Adenovirus encoding to β-galactosidase coupled to PMM2.1.2 nanoparticles or adenovirus alone were delivered locally to the adventitia of rabbit carotid arteries. Transfection rate was assessed histologically by the percentage of β-galactosidase positive cells in the vessel wall at 1 and at 2 weeks. There was significantly higher transfection rate when adenovirus was complexed with nanoparticles as compared to free adenovirus (At 1 week: 10±3.9% β-gal positive cells vs. 2.4±0.3% and at 2 weeks: 4.3±4.1% vs. 0%, P<0.005 for all). This difference was present in both the medial and adventitial layers. In conclusion, adenoviral mediated gene therapy was significantly enhanced by adsorbing the virus to PMM2.1.2 nanoparticles. This delivery method may be a good therapeutic strategy for the treatment of various vascular diseases.

The adult hair follicle: cradle for pluripotent neural crest stem cells.

This review focuses on the recent identification of two novel neural crest-derived cells in the adult mammalian hair follicle, pluripotent stem cells, and Merkel cells. Wnt1-cre/R26R compound transgenic mice, which in the periphery express beta-galactosidase in a neural crest-specific manner, were used to trace neural crest cells. Neural crest cells invade the facial epidermis as early as embryonic day 9.5. Neural crest-derived cells are present along the entire extent of the whisker follicle. This includes the bulge area, an epidermal niche for keratinocyte stem cells, as well as the matrix at the base of the hair follicle. We have determined by in vitro clonal analysis that the bulge area of the adult whisker follicle contains pluripotent neural crest stem cells. In culture, beta-galactosidase-positive cells emigrate from bulge explants, identifying them as neural crest-derived cells. When these cells are resuspended and grown in clonal culture, they give rise to colonies that contain multiple differentiated cell types, including neurons, Schwann cells, smooth muscle cells, pigment cells, chondrocytes, and possibly other types of cells. This result provides evidence for the pluripotentiality of the clone-forming cell. Serial cloning showed that bulge-derived neural crest cells undergo self-renewal, which identifies them as stem cells. Pluripotent neural crest cells are also localized in the back skin hair of adult mice. The bulge area of the whisker follicle is surrounded by numerous Merkel cells, which together with innervating nerve endings form slowly adapting mechanoreceptors that transduce steady skin indentation. Merkel cells express beta-galactosidase in double transgenic mice, which confirms their neural crest origin. Taken together, our data indicate that the epidermis of the adult hair follicle contains pluripotent neural crest stem cells, termed epidermal neural crest stem cells (eNCSCs), and one newly identified neural crest derivative, the Merkel cell. The intrinsic high degree of plasticity of eNCSCs and the fact that they are easily accessible in the skin make them attractive candidates for diverse autologous cell therapy strategies.

Changes in oligodendrocytes and myelin gene expression after radiation in the rodent spinal cord

Purpose The aim of this study was to assess changes in oligodendrocytes (OL), myelin gene expression, and their relationships with late demyelination after irradiation. Methods and materials Adult rats were given single doses of 8 or 22 Gy to the cervical spinal cord. Immunohistochemistry for APC or GST-π was used to identify OL. Changes in myelin gene expression were assessed using RT-PCR for proteolipid protein (PLP). Luxol fast blue staining was used to assess demyelination. CNP-βgeo transgenic mice were used to confirm some of the results of the rat model. Cells of the oligodendroglial lineage in these animals express β-galactosidase (β-gal). Results Early apoptosis of APC, GST-π, and β-galactosidase positive cells was observed in the spinal cord of rats and CNP-βgeo mice. At 24 h after 22 Gy, there was a significant decrease in OL density. Cell density continued to decline thereafter after both 8 and 22 Gy, and a reduction in PLP expression was observed at 4–5 weeks. A further decrease in PLP expression was seen beginning at 18 weeks after 22 Gy only. Demyelination was observed at 19 weeks after 22 Gy. Conclusions Apoptosis of OL and changes in OL density and PLP gene expression were observed early after both 8 and 22 Gy. This suggests that these early changes are unlikely to be directly related to the late demyelination observed.

Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells ,

Age-related decrease in bone formation is well described. However, the cellular causes are not known. Thus, we have established cultures of bone marrow stromal cells (MSC) from young (aged 18–29 years, n = 6) and old (aged 68–81 years, n = 5) donors. MSC were serially passaged until reaching maximal life span. Cell growth, markers of cellular senescence, and osteogenic and adipogenic potential were determined in early-passage and late-passage cells established from young and old donors. MSC from old donors exhibited a decreased maximal life span compared with cells from young donors (24 ± 11 population doublings [PD] vs 41 ± 10 PD, P < 0.05) and mean PD rate was lower in old donor cells (0.05 ± 0.02 PD/day) compared with young donor cells (0.09 ± 0.02 PD/day) ( P < 0.05). No differences were detected in number of senescence-associated β-galactosidase positive (SA β-gal +) cells and mean telomere length in early-passage cells obtained from young and old donors. However, MSC from old donors exhibited accelerated senescence evidenced by increased number of SA β-gal + cells per PD as compared with young (4% per PD vs 0.4% per PD, respectively). MSC from young and old donors were able to form similar amounts of mineralized matrix in vitro and of normal lamellar bone in vivo. In adipogenic medium similar numbers of adipocytes formed in cultures of young and old donors. In conclusion, aging is associated with decreased proliferative capacity of osteoprogenitor cells, suggesting that decreased osteoblastic cell number, and not function, leads to age-related decrease in bone formation.

D-galactose induces replicative senescence of rat pulmonary microvascular endothelial cells

To explore the relationship between senescent pulmonary microvascular endothelial cells (PMVECs) induced by D-galactose (D-gal) and apoptosis, PMVECs were cultured with DMEM containing D-gal (10 g/L) and identified by the following methods. We found that about 90% senescent cells were β-galactosidase positive cells, and 90% cells entered an irreversible G1 growth arrest. The cells in S and G2/M phases nearly disappeared. These results indicated that PMVECs ageing model induced by D-gal was established successfully. Compared with the control, PMVECs induced by D-gal showed that chromatin condensation and apoptotic bodies in nucleus were detected by fluorescence microscopy and transmission electron microscopy. Apoptotic cells in the early and middle phases increased significantly (39.8 ±2.8)% vs. (14.0 ±3.7)%. Typical Sub-G1 peak in the late apoptosis phase appeared in the terminal stage of PMVECs ageing. It is concluded that D-gal induces cultured PMVECs replicative senescence. Senescent PMVECs induced by D-gal were sensitive to apoptosis. Apoptosis is the regular feature of senescent PMVECs.

Demonstration of direct lineage between hepatocytes and hepatocellular carcinoma in diethylnitrosamine-treated rats

The question whether hepatocellular carcinoma (HCC) arises from dedifferentiation of mature hepatocytes or from proliferation of liver stem cells is still debated. In the present study, we used retroviral-mediated genetic labeling to investigate the fate of mature hepatocytes in rats after administration of diethylnitrosamine (DEN). Mature hepatocytes were genetically labeled by intravenous injection of retroviral vectors containing the Escherichia coli β-galactosidase gene coupled to a nuclear localization signal ( nls-LacZ) 1 day after partial hepatectomy. Liver biopsies performed after completion of hepatic regeneration showed that 18.3% of hepatocytes expressed the nls-LacZ transgene. Rats were then treated with DEN in drinking water for 12 weeks and sacrificed between 98 and 151 days after the onset of DEN administration. Clones of β-galactosidase positive cells were observed, half of which (53%) also expressed the placental form of glutathione- S-transferase (GSTp), a marker of preneoplastic cells. HCCs of various sizes expressing GSTp were present in all animals. Careful examination of 90 HCCs revealed that 16 (17.7%) also expressed nls-LacZ. This figure precisely matched the proportion of labeled hepatocytes before DEN treatment (18.3%). In conclusion, a random clonal origin of HCC from mature hepatocytes is seen in the DEN model of hepatocarcinogenesis. (H EPATOLOGY 2002;36:623-630.)

Functional Antagonism between Msx2 and CCAAT/Enhancer-binding Protein α in Regulating the Mouse Amelogenin Gene Expression Is Mediated by Protein-Protein Interaction

Ameloblast-specific amelogenin gene expression is spatiotemporally regulated during tooth development. In a previous study, the CCAAT/enhancer-binding protein alpha (C/EBPalpha) was identified as a transcriptional activator of the mouse amelogenin gene in a cell type-specific manner. Here, Msx2 is shown to repress the promoter activity of amelogenin-promoter reporter constructs independent of its intrinsic DNA binding activity. In transient cotransfection assays, Msx2 and C/EBPalpha antagonize each other in regulating the expression of the mouse amelogenin gene. Electrophoresis mobility shift assays demonstrate that Msx2 interferes with the binding of C/EBPalpha to its cognate site in the mouse amelogenin minimal promoter, although Msx2 itself does not bind to the same promoter fragment. Protein-protein interaction between Msx2 and C/EBPalpha is identified with co-immunoprecipitation analyses. Functional antagonism between Msx2 and C/EBPalpha is also observed on the stably transfected 2.2-kilobase mouse amelogenin promoter in ameloblast-like LS8 cells. Furthermore, the carboxyl-terminal residues 183-267 of Msx2 are required for protein-protein interaction, whereas the amino-terminal residues 2-97 of Msx2 play a less critical role. Among three family members tested (C/EBPalpha, -beta, and -gamma), Msx2 preferentially interacts with C/EBPalpha. Taken together, these data indicate that protein-protein interaction rather than competition for overlapping binding sites results in the functional antagonism between Msx2 and C/EBPalpha in regulating the mouse amelogenin gene expression.

Retroviral Labeling is an Appropriate Marker for Dividing Cells

5-Bromo-2′-deoxyuridne (BrdU) and 3H-thymidine label mitotically active cells, but they do not adequately mark the progeny of dividing cells for long term study. An alternative method is to label cells using the replication-defective CXL retroviral vector, which carries the lacZ gene encoding β-galactosidase; however, the ability of the CXL retroviral vector to pulse-label mitotically active cells selectively is not known. Cultures of proliferating muscle cells were simultaneously incubated with the CXL retrovirus and BrdU (10 μM) for 2 hr. After removing the retrovirus containing medium, the cells were maintained for an additional 24 hr in vitro before they were stained to detect β-galactosidase and BrdU simultaneously. More than 95% of β-galactosidase positive cells were also BrdU positive suggesting that the majority of β-galactosidase positive cells were in the S-phase of the cell cycle at the time of CXL retroviral administration. Therefore, the CXL retroviral vector is an appropriate pulse marker for dividing cells, and it is useful when it is desirable to know the fate of the progeny of a particular cell following a mitotic event.

The effect of passage number on fibroblast cellular senescence in patients with chronic venous insufficiency with and without ulcer

Background: Fibroblasts (fb) cultured from venous ulcer patients and patients with venous reflux disease without ulcer demonstrate characteristics of cellular senescence, such as increased fibronectin level and senescence-associated β-galactosidase (SA β-gal) positive cells. Cellular senescence is an in vitro event characterized by the progressive loss of proliferative capacity with increased passage number, and has been associated with impaired healing in vivo. This report examines progressive stages of cellular senescence in fb from the distal area (du-fb) and proximal fb (pu-fb) of patients with venous ulcer, as well as in distal fb (dr-fb) and proximal fb (pr-fb) from patients with venous reflux without ulcer, by comparing the population doubling time (T) and percent SA β-gal expression. Results: The mean value of T over 6 passages for fb in the ulcer group was 132.5 ± 29.0 hours for pu-fb and 492.9 ± 146.2 hours for du-fb ( P = 0.0009). For fb in the reflux group the mean value of T over 5 passages was 79.3 ± 12.8 hours for pr-fb and 94.2 ± 16.8 hours for dr-fb ( P = 0.8). Comparing ulcer and reflux fb, no difference in T was observed between pu-fb and pr-fb ( P = 0.6), but a difference was noted between du-fb and dr-fb ( P = 0.0004). The mean percent SA β-gal activity for fb in the ulcer group was 11.2% ± 3.1% for pu-fb and 63.8% ± 8.9% for du-fb ( P = 0.0001). Individual passages demonstrated significant difference ( P <0.05) in SA β-gal activity between pu-fb and du-fb at early and late passages. No difference was noted in SA β-gal activity for fb in the reflux group or between pu-fb and pr-fb, but comparison between du-fb and dr-fb was significant (63.8% ± 8.9% versus 7.8% ± 2.9%; P = 0.0001). Conclusions: The in vitro passage of du-fb and pu-fb in chronic venous ulcer patients has an effect on T and cellular senescence as measured by SA β-gal activity. Our data further suggest that du-fb are at a more progressive stage of cellular senescence when compared with pu-fb, and more importantly with fb cultured from patients with venous reflux without ulcer. These findings are consistent with impaired wound healing of venous stasis ulcer. The accumulation of senescent fb and a more advanced stage of cellular senescence of du-fb may explain why repeated episodes of venous ulceration are resistant to conservative treatment and require more aggressive measures of therapy.

Absence of toxicity associated with adenoviral-mediated transfer of the β-galactosidase reporter gene to neonatal rat islets in vitro

Transfer of genes with potential therapeutic utility to the pancreatic islets of Langerhans may enhance graft survival after islet transplantation. The aim of this study was to determine the optimal conditions for adenoviral-mediated gene transfer to the islets of Langerhans in the absence of vector-induced toxicity. Neonatal rat islets were transduced in groups of 25 with an adenoviral vector encoding β-galactosidase (AdβGal) at doses of MOI 0, 10, 100 and 1000 pfu per islet cell. All experiments were performed in triplicate. Efficiency of gene transfer was determined by gross inspection and estimation of the percentage of β-galactosidase positive cells after islet dispersion at 1, 4, 7 and 10 days post-transduction. Islet toxicity was assessed by measuring accumulated insulin levels at each time-point and by assessing static incubation insulin release at 3 and 10 days. Efficient dose-dependent gene transfer to the islets was documented at 1, 4, 7 and 10 days post-transduction. Transgene expression was relatively stable for the duration of the experiment. Insulin accumulation did not differ between transduced and non-transduced islets at each timepoint. Likewise, the insulin secretory response to glucose, obtained by dividing the insulin response to high glucose incubation by the insulin response to low glucose incubation was similar in transduced and non-transduced islets at 3 and 10 days at all doses studied. In summary, adenoviral-mediated transduction of islets results in dose dependent efficient gene transfer with relatively stable transgene expression in the absence of toxicity. This technology may be useful in the study of islet biology and also in the future in gene therapy approaches to the treatment of diabetes mellitus.

Fibroblasts cultured from distal lower extremities in patients with venous reflux display cellular characteristics of senescence

Purpose: Venous reflux precedes the development of venous ulcers. Our earlier work showed that the fibroblasts that are cultured from these wounds display more characteristics of senescence. We evaluated fibroblast senescence in patients with venous reflux but without ulcers to further investigate the role of venous reflux in the predisposition to venous ulcers. Methods: Fibroblasts that were isolated from skin biopsy specimens of the “gaiter” area (distal) and of the ipsilateral thigh of the same patient (proximal) were compared. Twelve patients with venous reflux (9 patients in clinical, etiologic, anatomic, and pathologic classification 4; 3 patients in classification 5) with an average venous filling index of 5.45 mL/s and 4 patients without venous reflux were enrolled in the study. The growth rates, the response to basic fibroblast growth factor (b-FGF), and the senescence markers (β-galactosidase activity at a pH level of 6, unstimulated fibroblasts fibronectin protein, and messenger RNA levels) were determined for each cell population. Results: The number of senescence-associated β-galactosidase positive cells (8.3% ± 1.9% vs 2.2% ± 0.8%; P = .008) and the level of cellular fibronectin protein (455.7 ± 80 vs 210 ± 51; P = .04) and messenger RNA (16.8 ± 6.8 vs 13.5 ± 5.7; P = .042) were significantly higher in the distal fibroblasts as compared with the proximal fibroblast cultures. The growth rates of the distal fibroblasts were lower when compared with the proximal fibroblasts (15,746 ± 4287 cells/day vs 29,550 ± 5035 cells/day; P < .002) but were not different in the presence of b-FGF (41,717 ± 9542 cells/day vs 47,030 ± 6133 cells/day; P = .53). In the patients without venous reflux, no site differences were noted in the growth rates or the senescence markers between the proximal and distal fibroblasts. Conclusion: Distal fibroblasts that are isolated from patients with venous reflux display more senescence characteristics than do proximal fibroblasts and have significantly lower growth rates. Despite senescence, b-FGF restored the distal-fibroblasts growth rate to that of the stimulated proximal fibroblasts, which proposes a therapeutic role for b-FGF. These changes precede ulcer formation and suggest a mechanism that is focal and intrinsically related to venous reflux. (J Vasc Surg 1998;28:1040-50.)

Evidence for keratinocyte stem cells in vitro: long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes.

Epidermis is renewed by a population of stem cells that have been defined in vivo by slow turnover, label retention, position in the epidermis, and enrichment in beta1 integrin, and in vitro by clonogenic growth, prolonged serial passage, and rapid adherence to extracellular matrix. The goal of this study is to determine whether clonogenic cells with long-term growth potential in vitro persist in vivo and give rise to a fully differentiated epidermis. Human keratinocytes were genetically labeled in culture by transduction with a retrovirus encoding the lacZ gene and grafted to athymic mice. Analysis of the cultures before grafting showed that 21.1-27.8% of clonogenic cells with the capacity for >30 generations were successfully transduced. In vivo, beta-galactosidase (beta-gal) positive cells participated in the formation of a fully differentiated epithelium and were detected throughout the 40-week postgraft period, initially as loosely scattered clusters and later as distinct vertical columns. Viable cells recovered from excised grafts were seeded at clonal densities and 23.3-33.3% of the colonies thus formed were beta-gal positive. In addition, no evidence of transgene inactivation was obtained: all keratinocyte colonies recovered from grafted tissue that were beta-gal negative also lacked the lacZ transgene. These results show that cells with long-term growth properties in vitro do indeed persist in vivo and form a fully differentiated epidermis, thereby exhibiting the properties of stem cells.

Efficient Transfer of Genes into Senescent Cells by Adenovirus Vectors via Highly Expressed αvβ5 Integrin

Although various methods for transferring genes into mammalian cells have been established, none have been successful with senescent cells. In this report, we present evidence of the efficient transfer of a gene into human senescent fibroblasts using an adenoviral vector. By employing a recombinant adenovirus vector harboring the β-galactosidase gene (AdCAGβNR), we observed a good correlation between the proportion of β-galactosidase positive cells and population doubling of the infected cells. In addition, 1.5- to 6.0-fold greater β-galactosidase activity was observed in senescent fibroblasts (population doubling [PD] = 58) than in young cells (PD=15). Western blotting analysis revealed that, compared with young fibroblasts, senescent fibroblasts expressed larger amounts of αvβ5 and αvβ3 integrins which were thought to form part of the adenovirus receptor. These results suggest that higher expression of αvβ5 and αvβ3 integrins in senescent cells renders them more sensitive to adenovirus infection than young cells. Thus, adenovirus vectors may prove to be useful in gene therapy strategies directed against senescence-related disorders.

Maximal Expression of Recombinant cDNAs in COS Cells for Use in Expression Cloning

In the process of establishing an expression cloning system for cell surface receptors we examined parameters which influence the expression of foreign genes in COS cells. The bacterial β-galactosidase gene was chosen as a reporter gene, since it permits the determination of (i) the fraction of cells transfected as well as (ii) the total activity of the synthesized enzyme in parallel experiments. This renders it possible to calculate the enzyme activity per individual cell. In transfected COS cells, the plasmid pXMgal directed a 20- and 10-fold higher β-galactosidase activity than pCH110 and pCDLgal, respectively. DEAE-dextran-mediated DNA uptake and protoplast fusion were found to result in higher expression rates than lipofection and electroporation. A coincubation of the cells with chloroquine during the DEAE-dextran transfection protocol caused, as reported, an increase of β-galactosidase positive cells but considerably reduced the total β-galactosidase activity. However, a 10% DMSO shock at the end of the transfection procedure simultaneously increased the number of transfected cells and the total β-galactosidase activity, thus maintaining the high expression per single cell. Using these optimized conditions, COS-1 cells expressed higher amounts of recombinant protein than COS-7 cells.

Liver-specific and position-effect expression of a retinol-binding protein- lacZ fusion gene (RBP- lacZ) in transgenic mice

Transgenic mice carrying a liver-specific promoter fused to a nuclear-targeted lacZ reporter gene were generated. Three separate lines of mice showed liver expression in the adult and no expression elsewhere in the animal. These results show that a 1.7 kb 5′-flanking region in the retinol-binding protein gene contains necessary and sufficient transcriptional signals for expression in adult livers. A fourth line (R197) did not express the transgene in the liver; instead, constitute lacZ expression was seen during postimplantation stages of development from Day 9.5 onwards and appeared to be associated with segmented structures including somites, branchial arches, and hindbrain rhombomeres until late fetal stages. The β-galactosidase positive cells in R197 were later seen to give rise to facial and flank musculature, and to other region-specific subpopulations of the jaws, neocortex, and brain stem. Northern blot analysis for the host retinol-binding protein RNA transcript did not show overlapping tissue expression with the reporter gene and suggests that transgenic phenotype seen in segmented embryonic structures of R197 and other extra-hepatic sites is from novel cis-acting transcriptional specificity. RNase protection assays of the R197 mRNA indicate that the lacZ sequences are appropriately transcribed downstream of the RBP canonical TATA box, even though the RBP promoter is itself silent. This result would suggest host flanking sequences with enhancer activity may have either activated the lacZ reporter gene or cooperated with the RBP promoter to create novel region-specific transcription. Breeding experiments have so far failed to produce offsprings that are homozygous for the transgene, and mating of transgenic F1 siblings routinely produce reduced litter sizes. Embryos that are homozygous for the transgene appear to be unable to survive beyond the egg cylinder stages of development. Thus, disruption of the host genome by insertional mutation appears to be manifest at an earlier stage than when position-effect expression of the transgene is first apparent. These experiments demonstrate that the component parts of a transgene may be subject to differential activation or suppression by host genomic flanking sequences and that even a strong, tissue-specific promoter may be overridden by host genes.