In vitro culture retards spontaneous activation of cell cycle progression and cortical granule exocytosis that normally occur in in vivo unfertilized mouse eggs

A microchip method has been developed for massive and parallel thermodynamic analyses of DNA duplexes. Fluorescently labeled oligonucleotides were hybridized with oligonucleotides immobilized in the 100 x 100 x 20 mum gel pads of the microchips. The equilibrium melting curves for all microchip duplexes were measured in real time in parallel for all microchip duplexes. Thermodynamic data for perfect and mismatched duplexes that were obtained using the microchip method directly correlated with data obtained in solution. Fluorescent labels or longer linkers between the gel and the oligonucleotides appeared to have no significant effect on duplex stability. Extending the immobilized oligonucleotides with a four-base mixture from the 3'-end or one or two universal bases (5-nitroindole) from the 3'- and/or 5'-end increased the stabilities of their duplexes. These extensions were applied to increase the stabilities of the duplexes formed with short oligonucleotides in microchips, to significantly lessen the differences in melting curves of the AT- and GC-rich duplexes, and to improve discrimination of perfect duplexes from those containing poorly recognized terminal mismatches. This study explored a way to increase the efficiency of sequencing by hybridization on oligonucleotide microchips.     

Sustained expression of high levels of human factor IX from human cells implanted within an immunoisolation device into athymic rodents

Immunoisolation of allogeneic cells within a membrane-bound device is a unique approach for gene therapy. We employed an immunoisolation device that protects allograft, but not xenograft, cells from destruction, to implant a human fibroblast line (MSU 1.2) in athymic rodents. Cells, transduced with the MFG-human factor IX retroviral vector, and expressing 0.9 microg/10(6) cells/day in vitro, were implanted in rats (four 40-microl devices, each containing 2 x 10(7) cells, two subcutaneously, two in epididymal fat) and in mice (two 20-microl devices, each containing 2 x 10(6) cells, subcutaneously). Plasma factor IX levels increased for 50 days, reaching maxima of 203 ng/ml (rat) and 597 ng/ml (mouse), and both continued at greater than 100 ng/ml for more than 140 days. A clone derived from the transduced cells, making 5 microg of factor IX/10(6) cells/day, was implanted within a device (one 20-microl device containing 2.5 x 10(6) cells), or without a device (1 x 10(7) cells implanted freely), either subcutaneously or in epididymal fat. The freely implanted cells expressed transiently, reaching more than 100 ng/ml in each site by day 4, but dropped to zero by day 20 (subcutaneous) or day 90 (epididymal fat). In devices, levels gradually increased to 100 ng/ml (subcutaneous) or 300 ng/ml (epididymal fat), remaining high for more than 100 days. These results show long-term, high-level expression of a human protein: (1) when cells are implanted within a cell transplantation device, but not when the cells are freely implanted, and (2) from a transgene driven by a viral promoter. An alloprotective device will enable the use of cloned cell lines that can be subjected to stringent quality control assessment that is impossible to achieve with autologous approaches.     

A controlled-release microchip

Much previous work in methods of achieving complex drug-release patterns has focused on pulsatile release from polymeric materials in response to specific stimuli, such as electric or magnetic fields, exposure to ultrasound, light or enzymes, and changes in pH or temperature. An alternative method for achieving pulsatile release involves using microfabrication technology to develop active devices that incorporate micrometre-scale pumps, valves and flow channels to deliver liquid solutions. Here we report a solid-state silicon microchip that can provide controlled release of single or multiple chemical substances on demand. The release mechanism is based on the electrochemical dissolution of thin anode membranes covering microreservoirs filled with chemicals in solid, liquid or gel form. We have conducted proof-of-principle release studies with a prototype microchip using gold and saline solution as a model electrode material and release medium, and we have demonstrated controlled, pulsatile release of chemical substances with this device.     

Inhibition of angiogenesis and growth of human nerve-sheath tumors by AGM-1470

The effectiveness of AGM-1470, a potent, fungal-derived inhibitor of angiogenesis, in suppressing the neovascularization and growth of human Schwann cell tumors was tested in six schwannomas, seven neurofibromas, and one neurofibrosarcoma. Tumor fragments from surgical specimens were implanted into the subrenal capsule of 348 nude mice (nu/nu). Seven days after implantation, the tumors were measured and vascularity was graded. The animals were then randomly assigned to one of two groups, to receive either saline (control group) or systemic AGM-1470 treatment. After 2 to 6 weeks of treatment, tumor size and degree of vascularity were recorded. In the six different schwannomas implanted into 138 mice, the average vascular grade in the control group after 2 weeks of treatment increased from 2.2 to 3.2 (+1.0), while in the AGM-1470-treated group it decreased from 2.2 to 1.7 (-0.5) (p < 0.01). In the seven different neurofibromas implanted into 158 mice, the change in the average vascular grade in control and AGM-1470-treated animals was +0.5 and -1.0, respectively (p < 0.01). In the one neurofibrosarcoma implanted into 52 mice, the change in average vascular grade in each group during the 6-week treatment period was +1.9 and -1.0, respectively (p < 0.01). Neurofibrosarcoma growth after 6 weeks of AGM-1470 treatment was only 8.5% of the growth found in the control animals (p < 0.01). This study determined that AGM-1470 is effective in inhibiting angiogenesis and the growth of human nerve-sheath tumors.     

Investigation on dimorphism of Blastomyces dermatitidis by agar-implantation method

By the agar-implantation developed by the authors the process of conversion on Blastomyces dermatitidis from mycelial phase to yeast phase was observed. First of all slide cultures of the fungus were prepared at room temperature. Upon confirmation of good hyphal growth, a cover glass was removed and a part of medium was cut out in a square of about 3 mm a side. After mice were laparotomied, each agar block cut out was implanted in the peritoneal cavity of mouse. The mice implanted with the agar blocks were killed, two each, every day for 14 days, and thereafter at intervals of a week for 2 months. Therefore, the implanted agar blocks were all recovered. They were examined directly by a light microscope with histopathological and electron microscopic examinations carried out at the same time. Within the peritoneal cavity of mouse, the intercalary and terminal chlamydospores were formed from hyphae. These subsequently swelled to become yeastlike cells and proliferated thereafter by budding.     

Utilization of energy for maintenance and for fat and lean gains by mice selected for rapid postweaning growth rate

The metabolizable energy intake (MEI) required for maintenance and the efficiency of utilization of metabolizable energy available for gain (MEA) were determined for a line of mice (rapid growth) selected for 41 generations for rapid postweaning weight gain and for a contemporarily mated line (control) that had been randomly selected. Feed intake of individually housed rapid growth and control males was restricted above maintenance or was ad libitum from 21 to 42 days of age. Regressions of change in body energy per unity metabolic body size on MEI per unit metablic body size showed that the maintenance requirement for each line of mice was 176 kcal per unit metabolic body size per day and that the rapid growth line was more efficient than the control line in utilizing MEA (50% vs. 23%) to promote an increase in body energy. Although the proportions of MEA used for fat (PF) and lean (PL) gains and the net efficiencies with which those proportions were utilized for fat (NF) and lean (NL) gains were unknown, the products of proportion and efficiency for fat gain (PF X NF or fat energy deposition coefficient) and for lean gain (PL X NL or lean energy deposition coefficient) were determined. The results demonstrate that 41 generations of selection for rapid postweaning weight gain did not change the lean energy deposition coefficient, but did alter the fat energy deposition coefficient. These data suggest that the two lines of mice use different proportions of MEA for fat gain and/or utilize MEA for fat gain at different efficiencies.     

Novel popout with nonsense strings: effects of predictability of string length and spatial location

The implanted system was composed of four silver ball electrodes placed in the burr hole of the skull, a reference placed subcutaneously near the nose, two electrodes for EMG, and a ground. The dural attachment was 0.1 mm in diameter. A cassette connector was placed on the back. Implanted cables between the cassette connector and all electrodes consist of twisted fine wires placed in a silicone tube 0.5 mm in diameter. The implanted electrode system weighed 0.8 g. The outlet cables were of the same materials used for implanted cables and placed in a silicone tube 1.5 mm in diameter. The impedance matching between these cables was successful and assured the minimum contamination of artifacts in the EEG recording of freely moving mice. Long-term (4-5 weeks) recording, thus, became possible without damage to the implanted materials. Monopolar recordings demonstrated the localized paroxysmal discharges during general tonic clonic convulsion in El mice. Several artifacts are presented.     

Correlation of changes in bone marrow cell sensitivity to the clastogenic effect of thiotepa and fertility in CBA/LacY mice in successive inbred generations

The causes of the significant increases in the sensitivity to of clastogenic effect of thio-TEPA in bone marrow cells of inbred CBA/LacY mice were studied. The increases were shown to be associated with decreased fertility. The fluctuations of fertility and the association of fertility and sensitivity with thio-TEPA were analyzed in foundation stocks (FSs) for 24 and 6 inbred generations, respectively. The stocks examined were obtained in the Laboratory of Experimental Biological Models and the Stolbovaya breeding facility. The correlation analysis revealed a significant association of fluctuations of the coefficient of fertility in two FSs (eta = 0.78 +/- 0.13) and a high negative correlation between fertility and mutagen sensitivity in inbred generations (r = -0.91 +/- 0.21). The comparison between the fluctuations of fertility for four generations revealed the same fluctuation period in CBA/LacY, DBA/2Y, and C57Bl/6JY mice. The detected phenomena were assumed to result from genetically determined regular fluctuations in genome functioning. This explained a number of observations and allowed several prognoses to be made.     

Levonorgestrel as a postcoital contraceptive

Time-release pellets of levonorgestrel (LNG), the progestogenic hormone contained in the contraceptive system Norplant, were implanted subdermally in mice, after the animals had mated and ovulated but before uterine implantation of embryos would have occurred, to examine whether the hormone could reduce the number of embryos that subsequently implanted and, if so, when it had to be administered in the postcoital period to achieve that effect. Hybrid female mice (C57BL x CBA) were paired with breeder males (CD-1) and LNG pellets were implanted on day 0, the day on which copulation plugs were found, or on day 2 or day 3 in the postcoital period. Mice in some groups were sacrificed on day 14 of the gestation period, and numbers of fetuses and/or resorption sites were counted, while mice in other groups were allowed to go to term. When LNG pellets were implanted subdermally on day 0 of the postcoital period, pellets designed to release 1.5 mg of hormone in 21 days failed to exhibit a contraceptive effect, but pellets designed to release 5 mg of hormone in 90 days were totally effective in preventing uterine implantation of embryos. Although the 5 mg pellets did not prevent embryos from implanting in all cases when administered on day 2, they prevented pregnancies from going to term by causing resorption of those embryos that did implant. When the pellets were implanted as late as day 3 in the postcoital period, uterine implantation of embryos occurred and fetuses were carried to term. Results of the study indicate that subdermal implants of LNG inserted postcoitally can prevent uterine implantation of embryos in mice, and thereby prevent pregnancy, despite fertilization of oocytes having occurred, if the hormone implants are inserted before day 3 of the postcoital period.     

Distribution and excretion of radiolabeled temoporfin in a murine tumor model

The biodistribution and excretion of temoporfin (tetra[m-hydroxyphenyl]chlorin, m-THPC), a recently developed photosensitizer, was investigated in BALB/c mice. [14C]temoporfin was administered intravenously (0.73 mumol/kg) to tumor-free mice or to mice implanted with the Colo 26 colorectal carcinoma. Blood, tissue and fecal samples were collected for 35 days and 10 days postdose from tumor-free mice and tumor-bearing mice, respectively. Blood concentrations fell rapidly such that at later time points they were indistinguishable from background counts. Tumor concentrations rose to a peak of 0.34 microgram temoporfin equivalents/mL at 2 days and then declined in parallel (log plot) with the blood concentrations. Tumor: tissue ratios at 2 days for skin, adipose tissue and skeletal muscle underlying the tumor were 1.5, 2.3 and 3.8, respectively. By 4 days the corresponding values were 1.6, 3.4 and 4.0. Nearly 40% of the administered radioactivity was excreted in the feces in the first 24 h and more than 80% had been excreted by 20 days. Less than 0.2% of the dose was recovered from the urine. An elimination half-life of 10-12 days was calculated from the excretion data.     

Transient cranial hemorrhage does not cause depressed contractility in cardiac neural crest-ablated chick embryos

The aim of this experiment was to study the osteogenesis in vivo of allogenic osteoblast combined culture with calcium phosphate composites. The osteoblasts were obtained by enzymatic digestion of periosteum from fibula subcultured to 13 generations, the cells were combined culture with hydroxyapatite and biphasic calcium phosphate. Subseguently, the composite was implanted into rabbits subcutaneously or intramuscularly. The blank material was implanted in the contralateral side as control. Four weeks later, all animals were sacrificed. All the implants were examined by gross observation, histological examination and EDXA. The results showed: 1. obvious ingrowth of connective tissue with very little inflammatory reaction; 2. new bone formation in the composites with deposit of Ca and P on the surface of osteoblast, but none in the blank materials; 3. no significant difference of new bone formation between the different sites of implantation or different materials, but those implanted intramuscularly had lamellae form of new bone while those implanted subcutaneously had only mineralization of extracellular matrix. The conclusion were: 1. the composites are biocompatible with prior osteogenesis property; 2. periosteal-derived allogenic osteoblasts obatined by enzymatic digestion could survive following implantation with bioactivity; 3. rich blood supply might be advantageous to new bone formation and its maturation.     

Characterization and screening for mutations of the growth arrest-specific 11 (GAS11) and C16orf3 genes at 16q24.3 in breast cancer

BACKGROUND: Both fibroblast-mediated cytokine gene therapy and bone marrow transplantation (BMT) have proven to be efficient protocols for the recovery of bone marrow depression. In this report, the effects of fibroblast-mediated interleukin (IL)-6 gene therapy, in combination with BMT, on the recovery of irradiation-induced bone marrow depression were investigated. METHODS: NIH3T3 fibroblast cells engineered to secrete IL-6 (NIH3T3-IL-6) or NIH3T3 cells transduced with the neomycin gene (NIH3T3-Neo), in combination with 10(7), 10(6), or 10(5) syngeneic bone marrow cells, were implanted into irradiated mice. RESULTS: The platelets and white blood cells in the peripheral blood of the irradiated mice increased greatly 12 days after implantation of NIH3T3-IL-6 cells and BMT, the white blood cell counts were restored to a normal level 32 days after the combined therapy, and the platelet number was obviously higher than that in mice implanted with NIH3T3-Neo and BMT. Twenty and 25 days after the combined therapy, the mice showed accelerated recovery of colony-forming unit (CFU)-granulocyte/macrophages and CFU-megakaryocytes when compared with the mice implanted with NIH3T3-Neo cells and BMT. Ten days after lethal irradiation with gamma rays, the spleens formed more CFU-spleen in mice implanted with NIH3T3-IL-6 cells and BMT than in mice injected with phosphate-buffered saline or NIH3T3-Neo cells. Combined therapy with NIH3T3-IL-6 cell implantation and BMT delayed the survival period of the hematopoietic-depressed mice significantly when compared with therapy with NIH3T3-Neo cell implantation and BMT. CONCLUSIONS: These data demonstrated that the combined therapy of fibroblast-mediated IL-6 gene therapy and BMT could significantly promote the recovery of irradiation-induced hematopoietic depression.     

Gastroesophageal reflux. Treatment by laparoscopy. 940 cases--French experience

The youngest (rings and young trophozoites) erythrocytic stages of Plasmodium yoelii nigeriensis and P. yoelii killicki, two rodent malaria subspecies developing very asynchronously in the blood, were separated from the other stages using a discontinuous Percoll-glucose gradient. They were inoculated into mice and the subsequent infection remained synchronous for two generations. The duration of the asexual cycle was found to be 18 h.     

Optimization of an assay for baculovirus titer and design of regimens for the synchronous infection of insect cells

Electrochemical treatment (ECT) of cancer utilizes direct current to produce chemical changes in tumors. ECT has been suggested as an effective alternative local cancer therapy. However, a methodology is not established, and mechanisms are not well studied. In vivo studies were conducted to evaluate the effectiveness of ECT on animal tumor models. Radiation-induced fibrosarcomas were implanted subcutaneously in 157 female C3H/HeJ mice. Larger rat fibrosarcomas were implanted on 34 female Fisher 344 rats. When the spheroidal tumors reached 10 mm in the mice, two to five platinum electrodes were inserted into the tumors at various spacings and orientations. Ten rats in a pilot group were treated when their ellipsoidal tumors were about 25 mm long; electrode insertion was similar to the later part of the mouse study, i.e., two at the base and two at the center. A second group of 24 rats was treated with six or seven electrodes when their tumors were about 20 mm long; all electrodes were inserted at the tumor base. Of the 24 rats, 12 of these were treated once, 10 were treated twice. and 2 were treated thrice. All treated tumors showed necrosis and regression for both mice and rats; however, later tumor recurrence reduced long-term survival. When multiple treatments were implemented, the best 3 month mouse tumor cure rate was 59.3%, and the best 6 month rat tumor cure rate was 75.0%. These preliminary results indicate that ECT is effective on the radiation-induced fibrosarcoma (RIF-1) mouse tumor and rat fibrosarcoma. The effectiveness is dependent on electrode placement and dosage.     

Bidirectional transfer between electrical and flurothyl kindling in mice: evidence for common processes in epileptogenesis

PURPOSE: This study sought to determine whether there was a transfer of seizure susceptibility between two models of epileptogenesis, electrical kindling and a newly described model of flurothyl kindling. In this study, we determined the effects of preexposure to one kindling agent on the seizure responsiveness to the other. METHODS: Mice were divided into three groups: (a) six mice (FLK) were kindled with flurothyl, rechallenged with flurothyl after a 28-day incubation phase, implanted with olfactory bulb (OB) electrodes, and electrically kindled; (b) six mice (ELK) were implanted with OB electrodes, electrically kindled to six stage 5 seizures, and given one flurothyl trial 3 days later and a second flurothyl trial after a 28-day incubation period; and (c) six mice (IMP) were implanted with OB electrodes, tested with flurothyl at the same times as the ELK group, and later electrically kindled. RESULTS: Mice that were previously kindled with flurothyl (FLK) had significantly faster electrical kindling rates to one stage 5 seizure or to six stage 5 seizures compared with animals in the ELK and IMP groups. Mice that were previously exposed to either electrical kindling or flurothyl kindling had significantly diminished latencies to generalized seizure onset (flurothyl-induced seizure thresholds) either before or after a 28-day incubation period compared with the IMP control mice. In addition, both the FLK and ELK groups had significantly increased percentages of mice expressing forebrain-brainstem seizures, compared with the IMP group, following either rechallenge with flurothyl after a 28-day incubation or focal electrical kindling. CONCLUSIONS: These findings indicate a near-complete bidirectional transfer between these electrical and flurothyl kindling models. Mice that were previously exposed to either electrical or flurothyl kindling have increased seizure susceptibilities and altered seizure phenotypes when exposed to the other seizure paradigm. Overall, these studies indicate that previous seizures are the critical determinant of the bidirectional transfer of seizure susceptibility observed, and not the electrical or pharmacologic properties of the original kindling agent. Finally, the observation of near identity in transfer characteristics between electrical and flurothyl kindling models suggests that the proepileptogenic processes initiated by exposure to either model are similar.     

Social health programs

During 1974--75, data on sex and family size were obtained from approximately 500 Brazilian college students and their parents. Sex ratios for the immediate and parental generations were 107:100 and 106:100, respectively. Correlation coefficients were calculated between sexes of various children in families of the immediate generation. A significant positive relationship was found between sexes of the first two children in families of two or more children. The overall correlation between sexes of successive births was positive but not significant. Correlation coefficients between sexes separated by one or more births were not significant. The observed frequencies of combinations of sexes within the various family sizes did not differ from expected frequencies. Average numbers of children per family were 4.39 and 6.80 for the immediate and parental generations, respectively. Average family sizes decreased as the formal educational level of the parents increased. If the parents' desired both sexes of children, such preferences had no measurable influence on family size.     

Induction of progestin receptors by estradiol in the forebrain of estrogen receptor-alpha gene-disrupted mice

Mice, rats, and humans have two types of estrogen receptors, estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta). Estrogen receptor-alpha gene-disrupted (ERalpha-disrupted) mice bear two nonfunctional copies of the ERalpha gene. This mutation blocks the synthesis of full-length ERalpha, renders the animals infertile, and inhibits the induction of female sexual behaviors by estradiol and progesterone. It is likely that many of the processes contributing to the regulation of sexual receptivity by estradiol and progesterone are compromised in ERalpha-disrupted mice. However, given the importance of progesterone in the regulation of sexual receptivity and given the importance of progestin receptors (PRs) in mediating the responses of females to progesterone, we investigated the effects of ERalpha disruption on the induction of PRs by estradiol in the forebrain. We hypothesized that estradiol would induce PRs in wild-type mice but not in ERalpha-disrupted mice. Ovariectomized wild-type and ERalpha-disrupted mice were implanted with either estradiol-filled capsules or empty capsules for 5 d, after which their brains were processed for the immunocytochemical detection of PR. Estradiol increased the number of PR-immunoreactive cells in both wild-type and ERalpha-disrupted mice. The residual responsiveness of ERalpha-disrupted mice to estradiol could be accounted for by an ERbeta-dependent mechanism or another as yet unidentified estrogen receptor; however, because ERalpha-immunoreactivity and PCR product representing the 3' end of ERalpha mRNA were found in at least one PR-containing region of the ERalpha-disrupted mice, an ERalpha splice variant may also mediate the induction of PR-immunoreactivity in ERalpha-disrupted mice.     

Intravenous self-administration of ethanol in beta-endorphin-deficient mice

Extensive research on both human alcoholics and in animal models of alcoholism has implicated the release of endogenous opioids in the consumption of ethanol. Various experiments using opioid antagonists have indicated that these drugs cause both humans and animals to decrease their consumption of ethanol. However, it remains unclear exactly which of the endogenous opioids mediates the rewarding effects of ethanol. The present experiment used intravenous self-administration of ethanol to determine whether beta-endorphin (BE)-deficient mice differed from wild-type (WT) mice in ethanol self-administration. The BE-deficient mice completely lack BE, but are otherwise similar to the WT mice. By using intravenous self-administration, we were able to rule out any ability of BE to mediate differences in ethanol consumption via palatability factors alone. Both types of mice were 7 generations backcrossed onto a C57BL/6J inbred strain background. During nine daily, 2-hr free-operant sessions, 14 BE-deficient and 17 WT mice could nosepoke for 75 mg/kg ethanol infusions delivered intravenously on an fixed-ratio 3 schedule with a 2-sec time-out after each reinforcer delivery. Reinforcer delivery occurred following nosepokes in only one of two holes. Contrary to what was expected, BE-deficient mice acquired selective operant responding for ethanol, whereas WT mice did not. Although the two genotypes did not differ in either operant or locomotor behavior during the first session, by the end of the nine sessions, BE deficient mice were reliably nosepoking for ethanol, whereas WT mice were not. These findings may indicate that BE is not essential for the postingestive reinforcing effects of ethanol in these animals.     

A pathogenic role of Th2 cells and their cytokine products on the pulmonary metastasis of murine B16 melanoma

The role of Th2 cells and the cytokines produced by these cells on experimental pulmonary metastasis of B16 melanoma was investigated in a murine model implanted with high metastatic (B16F10) or low metastatic (B16F1) melanoma cells. An average of 250 colonies of metastasis in the lungs was counted in mice (BF10 mice) at 14 days after the inoculation of 2 x 10(5) B16F10 cells/mouse, while <20 colonies were detected in mice (BF1 mice) inoculated with the same number of B16F1 cells. CD4+ CD11b+ TCR-alphabeta+ T cells (BF10-Th2 cells) were produced in the spleens of BF10 mice, while these cells were not detected in BF1 mice. The BF10-Th2 cells produced IL-4 and IL-10 into culture fluids when stimulated in vitro with anti-CD3 mAb. However, IL-2 and IFN-gamma were not produced. The level of a pulmonary metastasis in BF1 mice increased to the level observed in BF10 mice, when BF10-Th2 cells were adoptively transferred to BF1 mice. Also, an increase in the number of pulmonary melanoma was demonstrated in BF1 mice treated with 10 microg/kg murine rIL-4. The level of pulmonary metastasis in BF10 mice or in BF1 mice inoculated with BF10-Th2 cells decreased to the level observed in BF1 mice when mice were treated with an anti-IL-4 mAb at a dose of 250 microg/kg on days 1, 3, and 5 after tumor inoculation. These results suggest that the severity of pulmonary metastasis in mice receiving B16 melanoma cells is strongly influenced by the IL-4 released from tumor-associated Th2 cells.     

Pharmacologic control of a humanized gene therapy system implanted into nude mice

Systemic delivery of specific therapeutic proteins by a parenteral route of administration is a recognized practice in the management of several gene defects and acquired diseases. As an alternative to repetitive parenteral administration, gene therapy may provide a novel means for systemic delivery of therapeutic proteins while improving patient compliance and therapeutic efficacy. However, for gene therapy to be an efficacious and safe approach to the clinical management of such diseases, gene expression must be tightly regulated. These investigations demonstrate precise in vivo control of protein expression from cells that are engineered to secrete human growth hormone (hGH) in response to stimulation by rapamycin. The cells were implanted intramuscularly into nu/nu mice and stimulated by intravenous or oral administration of rapamycin. In vivo experiments demonstrate that the activity and pharmacokinetics of rapamycin determine the level of serum hGH that result from the engineered cells. In addition, responsiveness of the cells to rapamycin, number of cells implanted, hGH expression kinetics, and the pharmacokinetics of hGH itself, also influence the circulating levels of hGH after rapamycin stimulation. Controlled manipulation of several of these parameters, either independently or in combination, allows for precise regulation of circulating hGH concentration in vivo.