Regulation of expression of a sheep metallothionein 1a-sheep growth hormone fusion gene in transgenic mice.

Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.

Tandem high-dose therapy in rapid sequence for children with high-risk neuroblastoma.

PURPOSE: Advances in chemotherapy and supportive care have slowly improved survival rates for patients with high-risk neuroblastoma. The focus of many of these chemotherapeutic advances has been dose intensification. In this phase II trial involving children with advanced neuroblastoma, we used a program of induction chemotherapy followed by tandem high-dose, myeloablative treatments (high-dose therapy) with stem-cell rescue (HDT/SCR) in rapid sequence. PATIENTS AND METHODS: Patients underwent induction chemotherapy during which peripheral-blood stem and progenitor cells were collected and local control measures undertaken. Patients then received tandem courses of HDT/SCR, 4 to 6 weeks apart. Thirty-nine patients (age 1 to 12 years) were assessable, and 70 cycles of HDT/SCR were completed. RESULTS: Pheresis was possible in the case of all patients, despite their young ages, with an average of 7.2 x 10(6) CD34(+) cells/kg available to support each cycle. Engraftment was rapid; median time to neutrophil engraftment was 11 days. Four patients who completed the first HDT course did not complete the second, and there were three deaths due to toxicity. With a median follow-up of 22 months (from diagnosis), 26 of 39 patients remained event-free. The 3-year event-free survival rate for these patients was 58%. CONCLUSION: A tandem HDT/SCR regimen for high-risk neuroblastoma is a feasible treatment strategy for children and may improve disease-free survival.

Relationship between glucose and 20 alpha-hydroxysteroid dehydrogenase in fetal sheep erythrocytes.

We have examined whether glucose supply to fetal sheep erythrocytes limits the rate of 20 alpha-reduction of progesterone in blood and as such is associated with the progressive loss of 20 alpha-hydroxysteroid dehydrogenase activity which has been observed from 30 days before term. Enzyme activity in erythrocytes depleted of glucose by washing was regained in the presence of at least 0.167 mmol glucose/1. The cofactor NADPH was necessary to support the reaction in lysed cells. Addition of glucose to whole blood diluted 20-fold for assay of 20 alpha-hydroxysteroid dehydrogenase did not increase the rate of reaction. Infusion of dextrose to increase fetal plasma glucose concentrations had no effect on 20 alpha-hydroxysteroid dehydrogenase activity. Over the period from 114 to 137 days of gestation, both dextrose- and saline-infused fetuses showed a decline in enzyme activity from a combined mean of 1.45 +/- 0.21 (S.E.M.) to a mean of 0.78 +/- 0.18 mumol/ml erythrocytes per h. Fetal leucocytes did not contribute significantly to the activity of 20 alpha-hydroxysteroid dehydrogenase in whole blood. The rate of 20 alpha-reduction of progesterone in the blood of eight fetuses with indwelling carotid catheters declined from 2.31 +/- 0.09 mumol/ml erythrocytes per h at 90-95 days of gestation to 0.73 +/- 0.04 mumol/ml per h at 141-145 days. However, a consistent decline was only observed after 116-120 days. The apparent equilibrium position for progesterone reduction to 20 alpha-dihydroprogesterone varied between 83.9 +/- 1.8 and 65.7 +/- 4.2%.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of anaesthesia on ovarian follicular development and ovulation in the sheep subsequent to prostaglandin-induced luteolysis.

Prostaglandin analogues were used to induce luteal regression simultaneously in a number of ewes, thereby synchronizing the final stages of follicular maturation in these animals. Some of the ewes were anaesthetized for 24 h immediately after the injection of prostaglandin (experiment 1), and others for 15 h, starting 24 h after the injection of prostaglandin (experiment 2). In both experiments administration of anaesthetic significantly delayed the onset of oestrus and the time of ovulation relative to prostaglandin-treated control animals. The results from assays of blood samples collected at regular intervals in experiment 1 indicated that the preovulatory peak in the concentration of LH and the periovulatory changes in the concentration of FSH were similarly delayed and that during anaesthesia the level of LH was significantly reduced. It is suggested that the reduced level of LH, which probably resulted from a reduction in the secretion of releasing factor due to anaesthesia, failed to support oestrogen production by the Graafian follicle(s), thereby delaying the occurrence of oestrus and ovulation.

Evaluation of a rosette inhibition test for pregnancy diagnosis in pigs.

A rosette inhibition test was developed using pig lymphocytes and sheep red blood cells. Antilymphocyte serum (ALS) in the presence of complement inhibited rosette formation by greater than 95% at 1/250 declining to no inhibition at 1/8000. Sera obtained from a total of 14 pregnant sows before and 1, 2, 3 and 4 wk after mating were tested for their ability to augment the rosette depression caused by ALS. In one experiment in which the responses of 4 pregnant sows were compared to 4 non-pregnant sows by discriminant analysis, sera were classified correctly in 83% of the samples taken from either pregnant or non-pregnant sows. When the more usual method of calculating the rosette inhibition titre was used, the responses of sera from pregnant pigs were classified with 31% accuracy and those from non-pregnant pigs with 80% accuracy. In a second experiment, sera from 10 pregnant sows were classified with 25% accuracy using the rosette inhibition titre. Thus 4 of these pigs were classified as non-pregnant by this method. Data from the second experiment were not suitable for discriminant analysis. It was concluded that there was some factor present in the sera of pregnant pigs, particularly by 3 or 4 wk post-mating, which could be detected by the rosette inhibition test. However, the test is not sensitive enough to allow specific diagnosis of early pregnancy in pigs.

The commercial and agricultural applications of animal transgenesis.

The potential for commercial application of transgenic technologies in domestic animals is discussed in relation to the areas where a significant impact on agriculture might be expected. These are the endocrine system, novel biochemical pathways, structural proteins of milk and of textile fibers, and the immune system. Manipulation of the endocrine system has been investigated for some years and it is clear that very accurate control is needed over gene expression if this approach is to prove commercially useful. The area most advanced in commercial application is the production of high-value pharmaceutical proteins in the mammary glands of domestic animals. Other applications that are discussed remain to be proven in larger animals despite being demonstrated laboratory test animals. These include a functional cysteine biosynthetic pathway and a functional glyoxylate cycle transferred from bacteria to mice, and alterations to the proteins of hair that change the physical properties of the resultant fibers. Research is also actively directed toward novel approaches for providing domestic animals with resistance to insects.

Purification of 20 alpha-hydroxysteroid oxidoreductase from bovine fetal erythrocytes.

NADPH-dependent 20 alpha-hydroxysteroid oxidoreductase (20 alpha-HSD; EC 1.1.1.149) from bovine fetal erythrocytes was obtained for the first time free of hemoglobin by a new 2,500-fold purification scheme. This was achieved by a sequence of calcium phosphate gel absorption, ammonium sulfate fractionation, and affinity chromatography. The present results lead us to believe that the NADPH-dependent 3 beta-hydroxysteroid oxidoreductase activity, which was co-purified with 20 alpha-activity, may originate at the active site of 20 alpha-HSD (2).

Isolation of 3 beta,20 alpha-hydroxysteroid oxidoreductase from sheep fetal blood.

3 beta,20 alpha-Hydroxysteroid oxidoreductase has been isolated from ovine fetal blood by a 2,370-fold purification scheme of ammonium sulfate fractionation, calcium phosphate gel adsorption, affinity chromatography, and fast performance liquid chromatography. A new high performance liquid chromatography-based assay for measuring 20 alpha-reductase activity is described. The enzyme is a monomer with a molecular weight of 35,000 and uses NADPH as a cofactor for reductase activity. It reduces progesterone to 4-pregnen-20 alpha-ol-3-one or 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol with kinetic characteristics of Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1 or Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1, respectively. 5 alpha-Dihydrotestosterone competitively inhibits 20 alpha-reductase activity with a Ki value of 102 microM.

Cloning and sequencing of a cDNA encoding an ovine oestrus-associated oviducal protein.

Ovine oestrus-associated oviducal glycoprotein (oEGP) is synthesized and secreted specifically by the ampullary region of the ovine oviduct during the peri-ovulatory stages of the oestrous cycle. A cDNA that encodes oEGP was isolated and sequenced. Isolation of oEGP was achieved using the polymerase chain reaction (PCR) with primers based on a bovine oestrus-associated oviducal glycoprotein cDNA (bOGP) sequence. A 1599-bp cDNA encodes, in part, a deduced 519-amino acid sequence of mature protein which carries two potential N-linked glycosylation sites. The deduced amino acid sequence is more than 95% identical to that of bOGP and more than 74% identical to the first 491 amino acids of human oestrogen-dependent oviducal glycoprotein (hOGP). Northern blot hybridizations of RNA from several sheep tissues detected mRNA (2.4 kb) only in an ampulla oviduct sample.

Production of transgenic merino sheep by microinjection of ovine metallothionein-ovine growth hormone fusion genes.

Seven transgenic Merino sheep have been produced by the technique of pronuclear microinjection. Two different Sheep Metallothionein-1a-Sheep Growth Hormone fusion genes were used. Four of the transgenic sheep, all of which contained the gene MTsGH5, did not express the transgene. The remaining three sheep carrying the second fusion gene, MTsGH9, expressed the gene at high levels in a variety of tissues and had elevated blood levels of sheep growth hormone.

The potential of transgenic animals for improved agricultural productivity.

The techniques involved in the transfer of foreign DNA to domestic animals have advanced to the stage where transgenic animals that express foreign genes can be reliably produced, albeit still at low efficiency. This paper reviews the current status of some of the more important areas in agriculture where this technology is being applied. Numerous attempts have been made to modify the growth performance characteristics of domestic animals by the introduction of metallothionein/growth hormone fusion genes. A summary of our work with transgenic sheep is presented. The results demonstrate that the unregulated production of growth hormone in transgenic sheep reduces carcass fat, elevates metabolic rate and heat production, causes skeletal abnormalities and impairs survival. The introduction of new metabolic pathways to domestic animals offers an attractive approach to improved animal productivity. This paper summarises recent results of research directed towards the introduction of a cysteine biosynthetic pathway and the glyoxylate cycle to transgenic sheep. So far, the genes encoding the enzymes have been isolated and expressed both in cells in culture and in transgenic mice. The results of work currently in progress demonstrate that some modification of the fusion genes is required to enhance their expression in transgenic animals.

Ovarian response to PMSG and GnRH in ewes immunised against oestradiol-17 beta.

Forty-six adult merino ewes were immunised against oestradiol-17 beta-6 carbomethyloxime:human serum albumin and 48 comparable ewes were used as controls in an experiment to study the effects of gonadotrophin releasing hormone (GnRH) on ovulatory responses after treatment with pregnant mare's serum gonadotrophin (PMSG). All the ewes were treated with progestogen sponges for 14 days and received 1500 iu PMSG on the 12th day. Twenty-four control and 24 immunised ewes received 25 micrograms GnRH 21.5 hours and 23 hours after the sponges were withdrawn. Plasma samples were collected between 17 and 50 hours after the sponges were withdrawn and assayed for luteinising hormone (LH). Immunisation reduced the proportion of ewes which ovulated and their rate of ovulation. Injection of GnRH increased the proportion of immunised ewes ovulating (P less than 0.0005) and their rate of ovulation (P less than 0.0001). More unovulated follicles were observed in immunised ewes regardless of GnRH treatment (P less than 0.0001). The rate of recovery of eggs was reduced after immunisation. Treatment with GnRH produced a surge of LH of equal magnitude in the control and immunised ewes although not as many immunised ewes ovulated.

Termination of early pregnancy in ewes by use of a prostaglandin analogue and subsequent fertility.

Of 87 Dorset ewes injected at 20 to 60 days of pregnancy with either 125 micrograms or 250 micrograms of the prostaglandin F2 alpha analogue, cloprostenol, 72 (83%) were detected in oestrus by teaser rams within 7 days. A total of 60 ewes mated with fertile rams 14 to 28 days after treatment and 36 of these (60%) subsequently lambed. Thirty-eight ewes mated with fertile rams 29 to 56 days after treatment and 30 of these (79%) subsequently lambed. The difference in fertility between the 2 periods was not significant. Six additional ewes which did not respond to the cloprostenol lambed normally within 6 weeks. They were more than 100 days pregnant when treated. In ewes which first exhibited oestrus by 7 days of treatment, plasma progesterone concentrations fell from near 4 ng/ml to 0.6 ng/ml within 48 h of treatment. In ewes not detected in oestrus progesterone concentrations did not decrease to similar low levels (1.4 ng/ml; t-test p less than 0.005). Concentrations in the 6 ewes treated near 100 days of pregnancy dropped from 7.4 to 4.4 ng/ml over 48 h. Overall, the progesterone concentrations indicated that 92% of ewes treated at 20 to 60 days of pregnancy experienced rapid luteolysis in response to the cloprostenol. There were no differences between the 2 doses of cloprostenol in the responses or subsequent fertility of the ewes.

Decline with age in the rate of reduction of progesterone to 20 alpha-hydroxypregn-4-en-3-one in the blood of perinatal ruminants.

The activity of the enzyme 20 alpha-hydroxysteroid dehydrogenase present in erythrocytes of foetal and new-born ruminants has been determined by incubating 0.1 ml blood with 0.16 mumol [4-14C]-progesterone for 15 min at 39 degrees C in a final volume of 2 ml buffered saline. It was found that the activity, measured as mumol 20 alpha-hydroxypregn-4-en-3-one produced from progesterone per millilitre of erythrocytes per hour, declined from levels at birth as high as 1.50 mumol for sheep, 0.50 mumol for goats and 0.43 mumol for cattle to levels of around 0.11, 0.08 and 0.04 mumol respectively by 30-60 days of age. This decline in activity was also apparent in blood taken from sheep foetuses in which longitudinal studies were possible and appeared to have begun prior to 35 days before term. The highest activity obtained was 2.59 mumol for foetal sheep blood taken at 115 days of gestation. It is suggested that the observed decline in 20 alpha-hydroxysteroid dehydrogenase activity is a function of the replacement of foetal erythrocytes with adult-type erythrocytes which begins around 120 days of gestational age and that the role of the enzyme is to maintain an appropriate progestational environment within the foetoplacental unit.

The commercial and agricultural applications of animal transgenesis.

The commercial potential for transgenesis techniques is substantial, particularly in the fields of animal and plant agriculture. This results from productivity being a function of genetic potential and interaction with the environment, but environmental factors being only partially subject to influence by the farmer. Thus, concentrating on genotype improvement becomes an important goal if substantial cumulative gains in productivity are to be made. Historically, the genetic potential associated with important animal production traits, such as wool growth, milk yield, and body growth, has been improved by selective breeding, whereby phenotypically superior animals are used as parental stock for following generations. The high quality of the domestic animals in use in farming today compared with those of earlier centuries is witness to the success of the approach, but nevertheless, the method has significant limitations that have frustrated animal breeders for many years. The complex genetic interactions that combine to produce a particular animal phenotype result in slow genetic gain, averaging at best about 1-3% per year. In addition, separating a desired production trait from one or more undesirable traits is often very difficult. However, the most important of these limitations is the inability to transfer genetic information between species, because of the biological barrier that prevents interspecific breeding.