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Fusion genes combining the 5'-transcriptional regulatory region of the rat trypsin I gene and the structural gene of human growth hormone as a reporter were expressed to the high levels characteristic of the endogenous trypsin I gene selectively in the acinar cells of the pancreas of transgenic mice. As little as 232 base pairs of trypsin gene sequences containing the transcriptional start site and upstream promoter elements were sufficient to direct pancreatic expression. The tissue-specific expression was controlled transcriptionally. Trypsin-human growth hormone fusion transgenes also were expressed, although at low levels, in the stomach, an unexpected site for the expression of pancreatic digestive enzymes. Expression in the stomach of endogenous trypsin, elastase, and amylase genes in both normal and transgenic mice verified that transgene expression was consistent with normal expression of pancreatic genes. Endogenous amylase colocalizes with pepsinogen in the acinar cell-like Chief cells of the glandular portion of the mouse stomach. The expression of pancreatic genes in stomach cells is probably the consequence of similar developmental origins of pancreatic and gastric acinar cells from the primordial gut.

Transgenic sheep with elevated concentrations of circulating growth hormone (GH) were produced by microinjecting recombinant DNA into pronuclei of zygotes. The transgenes were fusion genes of non-GH promoters with coding sequences of various growth hormone genes including human, ovine or bovine. In addition, sheep transgenic with the human growth hormone releasing factor gene were produced. Non-GH promoters for fusion genes allowed novel regulation of GH production in ectopic tissues, including the kidney, liver and gut. Elevated levels of GH profoundly altered plasma IGF-1 without significantly altering rate of growth or feed efficiency. Carcass composition was altered with reduced fat. Elevated GH induced diabetes, resulting in death by 1 year of age. These studies indicate the need for improved regulation of inserted genes or investigation of alternative systems, such as GH receptors, to improve growth using the transgenic approach in ruminants.

We have produced transgenic pigs that harbour structural genes for bovine and human growth hormone (bGH and hGH) ligated to a mouse metallothionein-I (MT) promoter, human growth hormone-releasing factor (hGRF) ligated to the MT or mouse albumin (ALB) promoter, and human insulin-like growth factor-I (hIGF-I) ligated to MT promoter. From 0.31 to 1.03% of microinjected ova developed into transgenic pigs with the various fusion genes. Foreign GH was present in plasma of 61% of the MT-hGH and 89% of the MT-bGH transgenic pigs. Two of 7 pigs with MT-hGRF and all 3 ALB-hGRF transgenic pigs had high concentrations of GRF in their plasma, but plasma concentrations of porcine GH (pGH) were not higher in GRF transgenic pigs than in littermate control pigs. In contrast, plasma concentrations at birth ranged from 3 to 949 ng hGH/ml for MT-hGH transgenic pigs and 5 to 944 ng bGH/ml for MT-bGH transgenic pigs. Presence of the foreign GH depressed endogenous pGH to non-detectable levels. In MT-bGH transgenic pigs, plasma IGF-I was elevated more than 2-fold, plasma glucose was elevated about 30 mg/dl, and plasma insulin was 20-fold higher than in littermate or sibling control pigs. Two lines of pigs expressing the MT-bGH transgene gained 11.1% and 13.7% faster, and were 18% more efficient in converting feed to body weight gain than were sibling control pigs. Expression of the MT-bGH transgene caused a marked repartitioning of nutrients from subcutaneous fat into other carcass components, including muscle, skin, bone and certain organs. The persistent excess hGH or bGH in transgenic pigs was detrimental to general health; lameness, lethargy and gastric ulcers were the most prevalent problems. Gilts that expressed the hGH or bGH transgenes were anoestrous. Germ-line transmission was obtained in 4 of 5 expressing transgenic boars and 4 of 5 nonexpressing transgenic boars and gilts. From 2% to 73% of progeny inherited a transgene from founder transgenics. All transgenic progeny of MT-hGH, MT-bGH and MT-hGRF founder males expressed the transgene if their sire also expressed the gene. The concentration of bGH or hGH in plasma of transgenic progeny was similar to the concentration present in the founder transgenic.

A DNA fragment containing the promoter of the mouse metallothionein-I gene fused to the structural gene of rat growth hormone was microinjected into the pronuclei of fertilized mouse eggs. Of 21 mice that developed from these eggs, seven carried the fusion gene and six of these grew significantly larger than their littermates. Several of these transgenic mice had extraordinarily high levels of the fusion mRNA in their liver and growth hormone in their serum. This approach has implications for studying the biological effects of growth hormone, as a way to accelerate animal growth, as a model for gigantism, as a means of correcting genetic disease, and as a method of farming valuable gene products.

The development of methods for introducing foreign genes into the germ line of mice provides an approach for studying mechanisms underlying inducible and developmental gene regulation. Transgenic animals expressing foreign genes have thus been used to test models of the role played by specific DNA sequences in determining cell-specific expression. Results from these experiments suggest that tissue-specific expression is the consequence of a cis-acting regulatory sequence. However, these results do not exclude the possibility that cell-specific expression of some genes might be 'coded' by combinations of regulatory elements. We have previously described the production of transgenic mice from eggs microinjected with metallothionein-I/growth hormone (MGH) fusion genes, and now demonstrate that the juxtaposition of sequences from two different genes can be deciphered by cells to generate novel tissue specificities. Although expression of the endogenous metallothionein and growth hormone genes has not been detected in neuronal cells, transgenic mice clearly express an MGH fusion gene in a restricted subset of neurones. These results suggest a model in which tissue-specific patterns of expression of certain genes are determined by combinations of cis-acting regulatory sequences.

The promoter or regulatory region of the mouse gene for metallothionein-I was fused to the structural gene coding for human growth hormone. These fusion genes were introduced into mice by microinjection of fertilized eggs. Twenty-three (70 percent) of the mice that stably incorporated the fusion genes showed high concentrations of human growth hormone in their serum and grew significantly larger than control mice. Synthesis of human growth hormone was induced further by cadmium or zinc, which normally induce metallothionein gene expression. Transgenic mice that expressed human growth hormone also showed increased concentrations of insulin-like growth factor I in their serum. Histology of their pituitaries suggests dysfunction of the cells that normally synthesize growth hormone. The fusion genes were expressed in all tissues examined, but the ratio of human growth hormone messenger RNA to endogenous metallothionein-I messenger RNA varied among different tissues and different animals, suggesting that expression of the foreign genes is influenced by site of integration and tissue environment.

The dwarf little (lit) mouse is a model for the human hereditary disorder, isolated growth hormone (GH) deficiency type I. In these animals, dwarfism results from an autosomal recessively inherited gene mutation. The GH gene is present but production of GH mRNA is deficient, resulting in reduced serum GH and concomitantly decreased serum somatomedin. Growth retardation is evident by 15 days of age and adult animals reach approximately one-half normal size. Mutant mice of both sexes also exhibit a delayed onset of puberty, with males having a high degree of infertility. As administration of GH restores growth, we reasoned that growth failure in the mutant mice might be corrected by providing them with sufficient GH by gene therapy. Here we demonstrate that although the rat and human GH genes alone do not restore growth in transgenic mutants, a metallothionein-rat growth hormone fusion gene (MT-rGH) does. Moreover, the fertility of transgenic mutant males is improved; however, female fertility is impaired.

To study the role of insulin-like growth factor I (IGF-I) in mediating the growth-promoting function of GH, IGF-I expression was assayed in transgenic mice carrying either GH or GRF fusion genes. These mice exhibit enhanced growth as a result of high level ectopic expression of GH and have 2-fold elevation of both hepatic IGF-I mRNA levels and circulating IGF-I levels. Hepatic IGF-I mRNA levels are low at birth regardless of GH levels; they increase approximately 10-fold during postnatal development and become GH inducible 2 weeks after birth. The ontogeny of circulating IGF-I is similar. Accelerated growth in transgenic mice with GH fusion genes commences 3 weeks after birth despite high circulating GH levels at least as early as birth. In addition, endogenous mouse GH-secreting somatotroph cells in the pituitary are present in normal numbers at 3 weeks, but are undetectable 4 weeks after birth. Because the time at which IGF-I expression becomes GH responsive slightly precedes both the initiation of accelerated growth and the time when endogenous GH disappears, we conclude that IGF-I is directly involved in mediating the GH signal and that delayed induction of IGF-I gene expression is responsible, at least in part, for the delayed onset of other GH-responsive events.

Transfection of genes into tissue culture cell lines has demonstrated that relatively short DNA sequences can allow expression of immunoglobulin, insulin and chymotrypsin genes in their appropriate cell types. A definitive test of cell-specific gene expression, however, requires testing genes in every possible cell type, an experiment performed easily by introducing the gene in question into the germ line of an animal. Transfer of intact genes into mice has demonstrated that a mouse immunoglobulin kappa gene is expressed specifically in B lymphocytes, a rat elastase I gene is expressed specifically in pancreas and a chicken transferrin gene is expressed preferentially in liver. Mouse metallothionein-growth hormone fusion genes introduced into mice are preferentially expressed in the liver, consistent with the expression of endogenous metallothionein genes, but initial experiments with beta-globin genes have not revealed proper regulation. To identify the DNA elements required for pancreas-specific expression of the rat elastase I gene, we joined the 5'-flanking region of this gene to the human growth hormone (hGH) structural gene and introduced the fusion gene into mice. Here we demonstrate that a fusion gene containing only 213 base pairs (bp) of elastase I gene sequence directs expression of hGH in pancreatic acinar cells.

Transgenic pigs and sheep have been produced by the microinjection of single-cell zygotes and two-cell ova with linear molecules of mouse metallothionein I (MT) promoter/regulator fused to either the human growth hormone (hGH) or bovine growth hormone (bGH) structural genes. The foreign genes integrated into the chromosomes of 3 of 111 lambs or fetuses and 31 of 341 pigs or fetuses examined. Immunoreactive hGH or bGH was present in the plasma of two transgenic lambs and 19 transgenic pigs. The hGH concentration in plasma varied greatly among pigs and was unrelated to the number of gene copies that had integrated. Rate of growth was not enhanced in any of the transgenic pigs in comparison to their littermate controls. However, bGH and hGH exerted definite biological effects in transgenic pigs as evidenced by significantly depressed backfat measurements, elevated levels of insulin-like growth factor (IGF-I), stimulation of mammary development (by hGH) and reduction in porcine growth hormone (pGH) to nondetectable levels in plasma. Five of six founder transgenic pigs transmitted the MT-hGH gene construct to one or more progeny. Three progeny of a boar that expressed hGH also expressed the foreign gene.

Pronuclei of fertilized sheep ova were injected with fusion genes consisting of the mouse metallothionein-I promotor/regulator ligated to either the structural gene for bovine growth hormone (mMTbGH) or to a minigene for human growth hormone-releasing factor (mMThGRF). From a total of 842 sheep ova injected with mMTbGH and transferred into recipient ewes, 47 lambs were born. Two of the lambs were transgenic with mMTbGH, and both had bGH mRNA present in liver, kidney, and gut. In one lamb, plasma growth hormone was as high as 700 ng/ml. From a total of 435 sheep ova injected with mMThGRF and transferred to recipients, 54 lambs were born and 9 fetuses were collected. Nine of the 63 had integrated the mMThGRF gene. One of the nine had high concentrations of immunoassayable hGRF in its plasma and high variable plasma concentrations of ovine growth hormone. The lamb that expressed the hGRF gene did not release GH in response to an hGRF challenge. Four of five fetal offspring of a nonexpressing mMThGRF transgenic ram also contained the mMThGRF gene and, like the sire, failed to express the gene as determined by either liver hGRF mRNA or by plasma hGRF. Growth of the single transgenic lamb expressing hGRF was similar to control lambs. These studies demonstrate efficient introduction of genes into the sheep genome and indicate that transgenes are expressed and heritable.

Growth hormone (GH) appears to play a major role in a reciprocal axis that has been postulated between the thymus and pituitary glands. Our previous studies showed that thymic structure, as well as T-cell proliferation and IL-2 synthesis, could be restored in aged female Wistar-Furth rats by the implantation of GH3 pituitary adenoma cells. These cells secrete GH and some prolactin. We have now used three different approaches to determine whether GH affects a variety of immune events in vivo in both old and young rodents, and whether GH3 cells can directly affect progenitor T-cells in nude rats that congenitally lack a thymus gland. To test the effects of GH in aged rats, 750 micrograms of pituitary-derived ovine GH was injected 2 x daily into 26-month-old Fischer 344 rats for 5 weeks. This approach demonstrated that GH augments splenocyte proliferation to T-cell lectins as well as natural killer (NK) activity at low effector:target ratios even though morphologic characteristics of the thymus were not altered. To assess the effect of GH in young rodents, mice were studied that were transgenic for the rat metallothionein-GH gene. Histologic evaluation of thymus glands revealed that the amount of adipose tissue and the number of epithelial cells and Hassall's corpuscles are augmented in transgenic mice. Splenocyte proliferation at suboptimal mitogen doses is greater in transgenic than in control littermate mice, but neither IL-2 synthesis nor antibody synthesis to sheep erythrocytes is affected. The role of pituitary hormones on progenitor T-cells was then explored by implanting GH3 cells into Rowett nude rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrine profiles were examined in swine that had integrated and expressed a fusion gene consisting of mouse metallothionein-1 (MT) promoter fused to either a human (h) or bovine (b) GH structural gene. Eleven of 18 pigs that had integrated MT-hGH and eight of nine pigs that had integrated MT-bGH expressed the genes. The level of expression varied widely among pigs (14-4551 micrograms/l for MT-hGH and 23-1578 micrograms/l for MT-bGH). The level of expression varied over time within each pig with no general pattern. Concentrations of porcine GH (pGH) were lower in MT-hGH pigs that expressed the gene than in non-expressors or in littermate controls. Insulin-like growth factor-I (IGF-I) concentrations increased with age in all pigs and were raised threefold in pigs expressing either the MT-hGH or MT-bGH genes. Measurement of the foreign GH in samples taken at 15-min intervals failed to reveal any short-term fluctuations in concentration. Administration of hGH releasing factor (GRF) to pigs expressing MT-bGH resulted in attenuated release of pGH compared with that of contemporary controls. Concentrations of bGH did not change after GRF injection. Human and bovine GH expressed in transgenic pigs appear to be biologically active in that they induce IGF-I and suppress endogenous pGH secretion. The failure to find short-term fluctuations and the lack of response to GRF injections are consistent with a non-pituitary and non-GRF regulatable site of production.

To examine further the mechanism by which the synthesis of proteins translated on the rough endoplasmic reticulum is regulated in pancreatic beta-cells, the synthesis of growth hormone in islets from transgenic mice carrying the metallothionein-rat growth hormone gene fusion was studied. High glucose (17 mM) stimulated the synthesis and secretion of an apparently normally processed growth hormone. The stimulation of synthesis of growth hormone was less efficient than the stimulation of insulin synthesis in these islets, whereas the stimulation of release of labeled growth hormone paralleled that of insulin. These results are consistent with the hypothesis that signal recognition particle-mediated mechanism(s) may be involved in regulating the translational efficiency of secreted proteins in isolated islets (Welsh, M., Scherberg, N., Gilmore, R., and Steiner, D. F. (1986) Biochem. J. 235, 459-467). Furthermore, in the beta-cells, growth hormone follows the normal regulated pathway of secretory granule transport and exocytosis.

Serum levels of GH and insulin-like growth factor I (IGF-I) were genetically increased to investigate the physiological activities of these proteins. Lines of mice expressing chimeric genes composed of bovine GH, human GRF, or human IGF-I coding sequences fused to the mouse metallothionein I promoter were examined for consequences of chronic exposure to high levels of these peptides. Animals with elevated serum levels of GH (either bovine GH or mouse GH) have selective splanchnomegaly coupled with glomerular sclerosis and hepatocellularmegaly. Serum levels of insulin and cholesterol are increased. In contrast (with the exception of selective enlargement of organs), the chronic expression of IGF-I results in a different pattern of abnormalities. These findings suggest that the pathogenesis of GH-related disorders is not mediated solely by IGF-I.