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The tissue-specific expression of a fusion gene encoding the mouse metallothionein-1 promoter and the coding region of the human GH-releasing hormone (hGRH) gene was studied in transgenic mice by immunohistochemistry using an anti-hGRH serum that does not recognize endogenous mouse GRH. hGRH immunoreactivity (GRH-IR) was detected in specific cells of the pituitary, pancreas, kidney, duodenum, lung, testis, ovary, adrenal, heart, and brain. In the pituitary, using double immunofluorescent staining, GRH-IR was found in some, but not all, somatotrophs, gonadotrophs, thyrotrophs, and mammotrophs. GRH-IR was found in both pancreatic exocrine cells and endocrine islets. Within the islet, GRH-IR was colocalized in A and D cells with glucagon and somatostatin, respectively. Immunopositive cells in other tissues were localized in kidney proximal convoluted tubules, duodenal submucosal glands of Brunner, the smooth muscles of pulmonary arterioles, testicular Leydig cells, oocytes, adrenal medullary chromaffin cells, and cardiac atria. In the brain, GRH-IR was seen in the external layer of the median eminence and in perikarya and fibers of the hypothalamic arcuate nucleus, the parvocellular region of the paraventricular nucleus, the supraoptic nucleus, and the amygdala. Somatostatin-immunoreactive cell bodies and fibers in transgenic and control mouse hypothalamus were not appreciably different. In summary, hGRH expression in transgenic mice occurs in a cell-specific manner in the hypothalamus as well as in numerous other tissues, many of which have secretory functions.

Previous experiments have demonstrated that the human beta-globin gene is correctly regulated in transgenic mice. The beta-globin gene is not expressed in yolk sac-derived erythroid cells in early embryonic development but is expressed concomitantly with the adult mouse beta-globin genes in 14- to 16-day fetal liver and adult reticulocytes. In an attempt to localize sequences that direct erythroid-specific expression, fragments of the human beta-globin gene were inserted in the opposite orientation 200 base pairs (bp) upstream of an intact human A gamma marker gene, which is not expressed on its own in mouse fetal liver. In the experiments reported here, two beta-globin 3' sequences activated the marker gene specifically in fetal liver. One sequence is located in a 250-bp Pst I fragment 550-800 bp downstream from the poly(A) site; the other is located near an EcoRI site in the third exon. These two sequences are active individually, and their combined effect is greater than their effects alone. beta-Globin 5' sequences from -815 to -50 were also analyzed for activity in this assay. The 5' sequences did not activate the marker gene when tested alone but did stimulate expression that was already directed to adult erythroid tissue by the two 3' sequences. These results suggest that three separate sequences are involved in human beta-globin gene regulation. The two 3' sequences act as adult erythroid enhancers and the 5' sequence stimulates expression that is already determined to be erythroid specific.

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.

Class II genes of the MHC must be expressed by APC for activation of CD4+ T cells and efficient delivery of T cell help to B lymphocytes. Class II genes have restricted tissue expression and are under complex regulation. By using various deletion constructs of the class II E alpha d gene in transgenic mice we have mapped different 5' flanking regions which control E alpha d gene expression in distinct cell types. We demonstrate dissociate expression of E alpha d within the macrophage lineage as well as within the B cell lineage, and present evidence for a repressive element operative in B cells and macrophages. We describe the generation of novel transgenic lines with limited constitutive and inducible E alpha mRNA and I-E protein.

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.

Hybridomas were produced from spleen cells of kappa transgenic mice to investigate expression of the transgenic kappa gene, its effect on allelic exclusion and its effect on the control of light-chain gene rearrangement and expression. Our results show that the transgene is expressed normally and that the production of a complete immunoglobulin molecule turns off light-chain gene rearrangement.

Investigations were conducted to test the effects of alpha-amanitin on RNA synthesis in preimplantation mouse embryos. Exposure of embryos in culture to 1-100 microgram/ml alpha-amanitin produced a dose- and time-dependence suppression of total RNA synthesis as measured by incorporation of [3H]uridine. Synthesis of polyadenylated RNA in blastocyst-stage embryos was abolished by alpha-amanitin-treatment at concentrations and exposure times that suppressed total RNA synthesis by less than 15%. DNA-dependent RNA polymerase activity was measured in lysates of embryos at several stages of preimplantation development. alpha-Amanitin suppressed total polymerase activity assayed under ionic conditions favorable to the detection of RNA polymerase II. Electrophoretic analyses revealed that preincubation of blastocysts in 100 microgram/ml alpha-amanitin reduced labelling of cytoplasmic 28S and 18S RNA by inhibition of both synthesis and maturation of nucleolar 45SrRNA-precursor. This action of alpha-amanitin on nucleolar RNA synthesis cannot be correlated with the minimal suppression of nucleolar RNA polymerase activity and suggests that the synthesis and processing of rRNA may be under control of nucleoplasmic gene products.

A line of transgenic mice carrying a chimeric gene composed of human insulin-like growth factor I (IGF-I) coding sequences fused to the mouse metallothionein I promoter was generated to study the effects of chronically elevated exposure to IGF-I. Mice in this line overexpress IGF-I in most tissues studied and have circulating IGF-I levels 1.5 times the normal value. This results in a growth response manifested by a 1.3-fold increase in weight as a result of selective organomegaly without an apparent increase in skeletal growth. In addition, expression of the endogenous GH and IGF-I genes is inhibited. These results are consistent with IGF-I playing an important role in the control of somatic growth.

Analysis of 262 transgenic mouse pedigrees suggests that about 30% of the mice produced by microinjection of plasmids into pronuclei are mosaic in the germline. This implies that in these lines integration of the foreign DNA occurred after the first round of chromosomal DNA replication. In mosaics resulting from delayed integration the transgenic cells are usually distributed to both the trophectoderm and the inner cell mass, but sometimes to only one of these two cell types. Mosaicism of the inner cell mass results in even representation among the somatic tissues, and usually the germline as well; however, the germline is sometimes deficient in or entirely lacks transgenic cells. The germline precursor pool is distinct from the somatic precursor pools; apparently it is either determined prior to the primary germ layers or it is initially composed of fewer cells.

Transgenic mice were produced that carried in their germlines rearranged kappa and/or mu genes with V kappa and VH regions from the myeloma MOPC-167 kappa and H genes, which encode anti-PC antibody. The mu genes contain either a complete gene, including the membrane terminus (mu genes), or genes in which this terminus is deleted and only the secreted terminus remains (mu delta mem genes). The mu gene without membrane terminus is expressed at as high a level as the mu gene with the complete 3' end, suggesting that this terminus is not required for chromatin activation of the mu locus or for stability of the mRNA. The transgenes are expressed only in lymphoid organs. In contrast to our previous studies with MOPC-21 kappa transgenic mice, the mu transgene is transcribed in T lymphocytes as well as B lymphocytes. Thymocytes from mu and kappa mu transgenic mice display elevated levels of M-167 mu RNA and do not show elevated levels of kappa RNA, even though higher than normal levels of M-167 kappa RNA are detected in the spleen of these mice. Approximately 60% of thymocytes of mu transgenic mice produce cytoplasmic mu protein. However, despite a large amount of mu RNA of the membrane form, mu protein cannot be detected on the surface of T cells, perhaps because it cannot associate with T cell receptor alpha or beta chains. Mice with the complete mu transgene produce not only the mu transgenic mRNA but also considerably increased amounts of kappa RNA encoded by endogenous MOPC-167 like kappa genes. This suggests that B cells are selected by antigen (PC) if they coexpress the mu transgene and appropriate anti-PC endogenous kappa genes. Mice with the mu delta mem gene, however, do not express detectable levels of the endogenous MOPC-167 kappa mRNA. Like the complete mu transgene, the M-167 kappa transgene also causes amplification of endogenous MOPC-167 related immunoglobulins; mice with the kappa transgene have increased amounts of endogenous MOPC-167-like mu or alpha or gamma in the spleen, all of the secreted form. Implications for the regulation of immunoglobulin gene expression and B cell triggering are discussed.

The mouse homoeobox-containing genes exhibit temporally and spatially specific patterns of expression in embryonic and adult tissues and are thought to be important in regulation of development and cellular differentiation, perhaps by mechanisms analogous to homoeotic genes in Drosophila melanogaster. There has been no direct demonstration that expression of these mammalian genes can affect developmental processes, however. Hox-1.4, like other mouse homoeobox-containing genes, has been shown to be expressed in specific regions of the mid-gestation embryo, but is unique in that its highest level of expression in the adult animal is restricted to developing male germ cells. We have introduced a construct carrying the mouse Hox-1.4 gene into the germ line of mice to begin to identify the cis-acting elements required for proper expression and to assess the consequences of increasing Hox-1.4 gene expression. The construct was designed to produce normal Hox-1.4 protein from transcripts that are distinguishable from the products of the endogenous gene. The integrated transgene seemed to exhibit the appropriate tissue specificity of expression, but transcript levels were elevated in certain tissues, particularly the embryonic gut. This overexpression correlated with changes in the normal developmental program of the gut, resulting in an inherited abnormal phenotype known as megacolon.

Overproduction of the hepatitis B virus (HBV) large envelope polypeptide by transgenic mice containing the entire HBV envelope coding region leads to the formation of extremely long (up to 800 nm), occasionally branching, filamentous 22-nm-diameter hepatitis B surface antigen particles that accumulate within the endoplasmic reticulum of the hepatocyte and are not efficiently secreted. As the endoplasmic reticulum expands to accommodate the increasing cellular filament stores, the hepatocytes become enlarged, hydropic, and eosinophilic and also display the characteristic features of "ground-glass" cells. As filament storage progresses, the ground-glass cells undergo coagulative necrosis and the mice develop an age-dependent lesion, whose severity is related to the intracellular concentration of envelope polypeptide, that is characterized by focal hepatocellular degeneration and necrosis, lobular macrophagic inflammation, and increased serum transaminase activity. Advanced lesions demonstrate hepatocellular hyperplasia evident as lobular architectural disarray and microscopic hepatocellular nodules, many of which no longer contain detectable HBV envelope antigens. These changes may become extreme, producing a massively enlarged liver due to multifocal nodular regenerative hyperplasia. Overproduction of the large HBV envelope polypeptide exerts major structural constraints on HBV particle formation, leading to reduced secretion and progressive intracellular accumulation of hepatitis B surface antigen, which can reach sufficiently high concentrations to be directly cytotoxic to hepatocytes in this transgenic mouse system.

Transgenic mice that overproduce the hepatitis B virus large envelope polypeptide and accumulate toxic quantities of hepatitis B surface antigen (HBsAg) within the hepatocyte develop severe, prolonged hepatocellular injury that initiates a programmed response within the liver, characterized by inflammation, regenerative hyperplasia, transcriptional deregulation, and aneuploidy. This response inexorably progresses to neoplasia. The incidence of hepatocellular carcinoma in this model corresponds to the frequency, severity, and age of onset of liver cell injury, which itself corresponds to the intrahepatic concentration of HBsAg and is influenced by genetic background and sex. Thus, the inappropriate expression of a single structural viral gene is sufficient to cause malignant transformation in this model. These results suggest that severe, prolonged cellular injury induces a preneoplastic proliferative response that fosters secondary genetic events that program the cell for unrestrained growth.

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.

Class II major histocompatibility complex (MHC) gene expression in the mouse is generally limited to thymic epithelium and bone marrow-derived cells such as B lymphocytes and cells of the macrophage/dendritic cell lineage (M phi/DC). Class II-bearing B lymphocytes and M phi/DC possess antigen presenting cell (APC) function; that is, they can stimulate T lymphocytes reactive to either antigen plus MHC or foreign MHC alone. To assess whether non-bone-marrow-derived cells can acquire APC function and elicit graft rejection through expression of class II, we studied transgenic pancreatic islet beta cells that express a foreign class II (I-E) molecule. In vivo, grafts of I-E+ transgenic islets into I-E- naive hosts are not rejected unless the host is primed by an injection of I-E+ spleen cells. In vitro, the I-E+ beta cells are unable to stimulate T lymphocytes reactive to I-E plus a peptide antigen. Paradoxically, they induce antigen specific unresponsiveness in the T cells. We propose that expression of class II on non-lymphoid cells may serve as an extrathymic mechanism for maintaining self tolerance.

A method of deleting specific cell lineages has been developed that entails microinjection into fertilized eggs of a chimeric gene in which a cell-specific enhancer/promoter is used to drive the expression of a toxic gene product. We show that microinjection of a construct in which the elastase I promoter/enhancer is fused to a gene for diphtheria toxin A polypeptide results in birth of mice lacking a normal pancreas because of expression of the toxin in pancreatic acinar cells. A small pancreatic rudiment, containing islet and duct-like cells, was observed in some of the transgenic mice. This method provides a new approach for studying cell-lineage relationships and for analyzing cellular interactions during development.

We have shown recently that choroid plexus tumours frequently develop in transgenic mice which have developed from fertilized eggs injected with DNA molecules containing both simian virus 40 (SV40) early-region genes and metallothionein (MT) fusion genes, and several lines of mice have now been established in which all of the offspring that inherit the foreign DNA succumb to these tumours at 3-5 months of age (ref. 1 and our unpublished data). Several other tissues, notably thymus and kidney, occasionally also show pathological changes. SV40 large-T antigen protein and messenger RNA are always present in affected tissues at much greater concentrations than in unaffected tissues, suggesting that SV40 early-region genes are preferentially activated in choroid plexus, thymus and kidney and that this activation frequently leads to tumorigenesis in the choroid plexus. To determine which regions of the original constructs are important for this tumorigenesis, we have now tested several derivatives and report here that the large-T antigen is sufficient, that the MT fusion gene is dispensable and that the SV40 enhancer (72-base-pair repeat region) has an important role in directing tumours to the choroid plexus. Deletion of the SV40 enhancer region alone commonly leads to peripheral neuropathy, as well as liver and pancreatic tumours, which are the subject of the accompanying paper. Evidence is presented that these pathologies may result from an enhancing effect of the MT sequences on large-T antigen genes, made possible by removal of the otherwise dominant SV40 enhancer.

Protamines are abundant basic proteins involved in the condensation of sperm chromatin. In the mouse, protamine genes are transcribed postmeiotically in round spermatids. We have cloned and sequenced the mouse protamine 1 gene. Ten lines of transgenic mice harboring marked protamine 1 sequences were generated by microinjection of fertilized eggs. Transcription of the transgene is restricted to round spermatids and in several cases exceeds that of the endogenous gene. The cis-acting sequences required for tissue-specific protamine expression reside on a 2.4-kilobase restriction fragment. Prospects for using transgenic mice to address fundamental questions of male germ-cell development are discussed.

Erythroid-specific DNase I super-hypersensitive (HS) sites that are normally located far upstream of the human beta-globin locus were inserted immediately upstream of a 4.1-kb fragment containing the human beta-globin gene. These constructs (HS beta) and a construct containing the beta-globin gene alone (beta) were microinjected into fertilized mouse eggs, and expression was analyzed in erythroid fetal liver and brain of day-16 embryos that developed. Only 7 of 23 animals that contained the beta gene alone expressed human beta-globin mRNA in erythroid tissue, and the average level of expression per gene copy was 0.3% of endogenous mouse beta-globin mRNA. In contrast, 50 of 51 transgenic mice that contained various HS beta constructs expressed the transgene specifically in erythroid tissue. The average level of expression per gene copy for constructs containing all five upstream HS sites was 109% of endogenous mouse beta-globin mRNA. Constructs that contained a single super-hypersensitive site (HS II beta) expressed 40% as much human beta-globin as mouse beta-globin mRNA per gene copy. These results demonstrate that the HS VI site, normally located downstream of the human beta-globin locus, is not required for high-level expression. Furthermore, the results demonstrate that high levels of human beta-globin gene expression can be obtained in transgenic mice even when a relatively small fragment of DNA (1.9 kb) containing erythroid-specific super-hypersensitive site II (HS II) is inserted upstream of the human beta-globin gene.

We have introduced the class II E alpha d gene into (C57BL/6 X SJL) F2 mice which do not express their endogenous E alpha gene. The mRNA expression of the E alpha d gene shows the same tissue distribution as the endogenous class II genes except in the case of one mouse, which carried 19 copies of the E alpha d gene. In this mouse expression of E alpha d mRNA was seen in all tissues tested. Expression of the transgene was induced by gamma-interferon in isolated macrophages from the transgenic mice. In addition, fluorescence activated cell sorter (FACS) analysis, mixed lymphocyte response and antigen-presentation experiments showed that the product of the transferred gene is expressed on the cell surface and functions as a major histocompatibility complex restriction element. Transmission of the gene occurred only with female transgenic mice, all males were infertile or did not transmit the gene, suggesting an effect of the transferred DNA sequence on male reproductive function.

The pituitaries of transgenic mice that express a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin-14 exclusively in the gonadotrophic cells. The purpose of this study was to determine whether somatostatin expressed from the foreign fusion gene enters the normal secretory pathway within these cells. Immuno-gold labeling of serial thin sections localized somatostatin to the secretory granules of gonadotropin-producing cells. The gonadotroph-specific hypophysiotropic factor, luteinizing hormone-releasing hormone caused a dose-dependent secretion of somatostatin when applied to primary pituitary cultures from these mice. Growth hormone-releasing hormone, thyrotropin-releasing hormone, corticotropin releasing factor, and dopamine did not affect somatostatin secretion. These experiments demonstrate that a neurosecretory peptide encoded by a foreign gene can enter the regulated secretory pathway of pituitary cells from transgenic mice.

A complete, functional gamma 2b gene (pVCM) was cloned from a mouse hybridoma (VD93) with antibody activity to Pseudomonas aeruginosa. DNA sequencing of the VDJ region of pVCM determined that the VH gene was a member of the J558 family rearranged to JH2. Upon transfection into myeloma cells the gamma 2b gene gave rise to high levels of gamma 2b mRNA and gamma 2b protein. The gamma 2b protein had the same IEF pattern as the parent hybridoma protein VD93 and the antibodies formed from a combination of the pVCM gamma 2b chains and the myeloma lambda-chains bound weakly to P. aeruginosa. However, the hybrid antibodies did not discriminate between the serotypes 2 and 3, whereas the parent protein was specific for serotype 3. Transgenic mice were produced with the pVCM gamma 2b gene which expressed the gamma 2b mRNA (both membrane and secreted forms) only in lymphoid organs. However, contrary to expectations, the gamma 2b mRNA levels were higher in T cells than in B cells in three different transgenic lines. The serum of the transgenic mice had no activity to P. aeruginosa indicating the importance of L chains for the conformation of the Ag binding site. These gamma 2b transgenic mice provide a convenient tool for the study of feedback inhibition of Ig gene rearrangement.

The ability to introduce foreign DNA into the genome of mice offers unique opportunities to produce new models of disease process. Recent experiments have shown that integration and expression of simian virus 40 (SV40) T antigen genes and the murine mammary tumour virus (MMTV)-myc genes in transgenic mice can lead to the development of neoplasia in a remarkably tissue-specific manner. In the case of SV40-bearing mice, tumours consistently develop in the choroid plexus. In the accompanying paper, we show that the 72-base pair (bp) enhancer in the SV40 genome is instrumental in directing tumorigenesis to the choroid plexus. However, when the enhancer is deleted from a construction also containing the metallothionein-human growth hormone fusion gene (SV delta e-MGH), an entirely new pattern of pathology results. The present report focuses on transgenic mice carrying this construct; they develop demyelinating peripheral neuropathies, hepatocellular carcinomas and islet cell adenomas.

Teratocarcinoma cells (OTT 6050) from 129 SvSl C P mice were transferred into blastocysts from random bred Swiss albino mice. The blastocysts were placed in the uteri of foster mothers and the adults resulting from these blastocysts were studied for evidence of an effect of the transferred malignant cells. Sixty adults resulted from the experiments, and one of the adult mice that had received teratocarcinoma cells in the blastocyst stage showed several stripes of agouti hair. All the adult animals received grafts of skin from animals identical to those supplying the cells. The animals that resulted from blastocysts into which cells had been transferred maintained skin grafts for a significantly longer period than did controls. These experiments indicate that the transferred malignant cells were able to establish small colonies in the embryos and that some of these cells persisted into the adult.

The human alpha 1-globin gene was fused downstream of two erythroid-specific DNase I super-hypersensitive sites that are normally located upstream of the human beta-globin locus. This construct was injected into fertilized mouse eggs, and expression was analyzed in 16-day fetal livers and brains. All 11 fetuses that contained intact copies of the transgene expressed correctly initiated human alpha-globin mRNA in the erythroid fetal liver but not in brain. Levels of expression ranged from 4% to 337% of endogenous mouse beta-globin mRNA. A human alpha-globin construct that did not contain super-hypersensitive sites was not expressed. These results demonstrate that human beta-globin locus activation sequences can stimulate high levels of human alpha-globin gene expression in erythroid tissue of transgenic mice. The results also provide a foundation for experiments designed to coexpress human alpha- and beta-globin genes in transgenic mice and suggest a feasible approach for production of a mouse model for human sickle cell disease.

Direct microinjection has been used to introduce foreign DNA into a number of terminally differentiated cell types as well as embryos of several species including sea urchin, Candida elegans, Xenopus, Drosophila and mice. Various genes have been successfully introduced into mice including constructs consisting of the mouse metallothionein-I (MT) promoter/regulator region fused to either the rat or human growth hormone (hGH) structural genes. Transgenic mice harbouring such genes commonly exhibit high, metal-inducible levels of the fusion messenger RNA in several organs, substantial quantities of the foreign growth hormone in serum and enhanced growth. In addition, the gene is stably incorporated into the germ line, making the phenotype heritable. Because of the scientific importance and potential economic value of transgenic livestock containing foreign genes, we initiated studies on large animals by microinjecting the fusion gene, MT-hGH, into the pronuclei or nuclei of eggs from superovulated rabbits, sheep and pigs. We report here integration of the gene in all three species and expression of the gene in transgenic rabbits and pigs.

The mouse alpha-fetoprotein gene is activated in embryonic development in the visceral endoderm of the extraembryonic yolk sac and the fetal liver and gut. Transcription of the gene is subsequently repressed in the neonatal liver. To ask whether the DNA sequence elements required for tissue-specific activation are the same or different from those required for postnatal developmental regulation of the gene, modified copies of the alpha-fetoprotein gene were microinjected into fertilized mouse eggs. Those animals which developed to term and carried integrated copies of the modified gene were analyzed for expression. In approximately 50% of such animals, the introduced gene was active only in the three cell lineages which expressed the authentic alpha-fetoprotein gene. Furthermore, its expression was repressed in the neonatal liver. Thus, we conclude that the modified genes, which included either 7 or 14 kilobase pairs of 5'-flanking DNA, contained the DNA sequence information to direct both tissue-specific expression and developmental regulation. The observation that 50% of the mice which carried the modified gene did not express it in any tissue, combined with the fact that the level of expression was highly variable between expressing transgenic animals, suggested that the gene was susceptible to its site of integration in the mouse genome.

The outer membrane of the hepatitis B virus consists of host lipid and the hepatitis B virus major (p25, gp28), middle (gp33, gp36), and large (p39, gp42) envelope polypeptides. These polypeptides are encoded by a large open reading frame that contains three in-phase translation start codons and a shared termination signal. The influence of the large envelope polypeptide on the secretion of hepatitis B surface antigen (HBsAg) subviral particles in transgenic mice was examined. The major polypeptide is the dominant structural component of the HBsAg particles, which are readily secreted into the blood. A relative increase in production of the large envelope polypeptide compared with that of the major envelope polypeptide led to profound reduction of the HBsAg concentration in serum as a result of accumulation of both envelope polypeptides in a relatively insoluble compartment within the cell. We conclude that inhibition of HBsAg secretion is related to a hitherto unknown property of the pre-S-containing domain of the large envelope polypeptide.