Porcine growth hormone gene expression from viral promoters in transgenic swine

Abstract

The production of porcine growth hormone (pGH) from novel expression vectors containing the promoter/enhancer elements of the Moloney murine leukemia virus (MLV) LTR or the human cytomegalovirus (CMV) immediate early gene was examined in transgenic swine. Both fusion genes resulted in elevated levels of serum pGH, elevation of insulin‐like growth factor 1 (IGF‐1), and a pronounced decrease in carcass fat deposition. The two viral promoter/enhancer elements were constitutively active in the transgenic swine throughout the life of the animals. In individual swine, the CMV‐pGH transgene was expressed predominantly in the pancreas while the MLV‐pGH transgene was expressed in a wide variety of tissues. These swine were infertile, had insulin resistance, and demonstrated an accelerated form of osteochondritis dissicans. Our results show that excess pGH produces a phenotype identical to that seen in swine expressing heterologous growth hormones, and provides a baseline for assessing the overall efficiency of producing transgenic swine. Furthermore, our data suggests that unregulated pGH production, even at 15 times normal levels and independent of the tissue source, has adverse effects that outweigh the desired reduction in carcass fat deposition in transgenic swine.     

The methylation-free status of a housekeeping transgene is lost at high copy number

Transgenic mouse lines were established bearing tandem arrays of a fusion construct comprising the promoter region of a housekeeping gene, HMGCR, encoding 3-hydroxy 3-methylglutaryl CoA reductase, linked to a bacterial cat reporter gene encoding chloramphenicol acetyltransferase (CAT). CAT activity was observed in all transgenic mouse tissues examined. The methylation state of the fusion transgene was determined. In non-transgenic mice the endogenous HMGCR promoter is devoid of methylation while flanking regions are extensively modified. In HMGCR-cat transgenic mice the fusion gene promoter was found to be similarly hypomethylated. However, the extent of hypomethylation varied with copy number: methylation-free status was progressively lost with increasing transgene copy number. Further transgenic mouse lines were constructed carrying a truncated HMGCR regulatory region linked to cat. Transgene expression and hypomethylation were observed in testis but not in any other tissue, and testis-specific methylation-free status was also lost at high copy number. Loss of hypomethylation at high copy number may indicate that saturable DNA-binding factors normally protect the HMGCR promoter from methylation.     

Variegation and silencing in a lentiviral-based murine transgenic model

Lentiviral based constructs represent a recent development in the generation of transgenic animals. The ease of use, and the fact that the same backbone vectors can be used to down-modulate endogenous gene expression and to produce transgenic animals overexpressing a gene of interest, have fuelled growing interest in this technology. In this study, we have used a lentiviral delivery system to generate transgenic mice expressing altered levels (up or downregulated) of a gene of interest. Although this lentiviral-based approach led to high levels of transgenesis and germ line transmission, a wide variation in transgene expression was observed in most first and second generation mouse lines. In particular, despite the segregation of integrants into single-copy expressing mouse lines, transgene expression appeared to be the target of epigenetic regulatory mechanism, often causing the coexistence of high and low transgene expressing cells within a given tissue such as blood peripheral lymphocytes. The establishment and analysis of large number of mouse lines may therefore be required to select a stable transgenic line with pancellular expression of a gene of interest using this lentiviral-based approach.     

Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression

To study the frequency of germ-line transformation and to examine the reproducibility of tissue-specific transgene expression, we produced several lines of transgenic zebrafish expressing a recombinant chloramphenicol acetyltransferase (CAT) gene. Supercoiled plasmids containing both Rous sarcoma virus and SV-40 promoter sequences upstream of the CAT coding region were injected into zebrafish embryos prior to first cleavage. CAT activity could be detected in batches of injected embryos as early as 8 h and up to at least 12 days post-fertilization. Approximately 18% of injected fish raised to maturity exhibited CAT activity in their fins, and approximately 5% of injected fish became stable germ-line transformants. Breeding studies indicated that although transgenic founder fish were frequently germ-line mosaics, transgenic individuals of subsequent generations were fully hemizygous for the transgene marker. The transgenes present in the F1 progeny of four independent lines were relatively well expressed in fin and skin, while lower levels of expression were observed in heart, gill and muscle. Little or no CAT expression was observed in the brain, liver and gonad. A monoclonal antibody directed against the CAT gene product consistently revealed variegated patterns of CAT expression in ectodermally derived fin epidermal cells in three of these lines. These results show that it is possible to efficiently produce stable germ-line transformants of the zebrafish and to observe reproducible tissue-specific patterns of transgene expression in this organism. Possible mechanisms for the variegated expression observed within tissues are also considered.     

Activity of the adenosine deaminase promoter in transgenic mice.

The promoter of the human gene for adenosine deaminase (ADA) is extremely G/C-rich, contains several G/C-box motifs (GGGCGGG) and lacks any apparent TATA or CAAT boxes. These features are commonly found in promoters of genes that lack a strong tissue specificity, and are referred to as "housekeeping genes". Like other housekeeping genes, the ADA gene is expressed in all tissues. However, there is a considerable variation in the levels of expression of the ADA protein in different tissues. In order to study the activity of the ADA promoter, transgenic mice were generated that harbor a chimeric gene composed of the ADA promoter linked to a reporter gene encoding the bacterial enzyme Chloramphenicol Acetyl Transferase (CAT). These mice reproducibly showed CAT expression in all tissues examined, including the hemopoietic organs (spleen, thymus and bone marrow). However, examination of the actual cell types expressing the CAT gene revealed the ADA promoter to be inactive in the hemopoietic cells. This was substantiated by a transplantation experiment in which bone marrow from ADA-CAT transgenic mice was used to reconstitute the hemopoietic compartment of lethally irradiated mice. The engrafted recipients revealed strongly reduced CAT activity in their hemopoietic organs. The lack of expression in hemopoietic cells was further shown to be correlated with a hypermethylated state of the transgene. Combined, our data suggest that the ADA promoter sequences tested can direct expression in a wide variety of tissues as expected for a regular housekeeping gene promoter. However, the activity of the ADA promoter fragment did not reflect the tissue-specific variations in expression levels of the endogenous ADA gene. Additionally, regulatory elements are needed for expression in the hemopoietic cells.     

A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters

Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice.     

Establishment and characterization of CAG/EGFP transgenic rabbit line

Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP florescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.     

Expression and regulation of the human GLUT4/muscle-fat facilitative glucose transporter gene in transgenic mice.

To study the molecular basis of tissue-specific expression of the GLUT4/muscle-fat facilitative glucose transporter gene, we generated lines of transgenic mice carrying 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene (hGLUT4[2.4]-CAT). This reporter gene construct was specifically expressed in tissues that normally express GLUT4 mRNA, which include both brown and white adipose tissues as well as cardiac, skeletal, and smooth muscle. In contrast, CAT reporter activity was not detected in brain or liver, two tissues that do not express the GLUT4 gene. In addition, the relative levels of CAT mRNA driven by the human GLUT4 promoter in various tissues of these transgenic animals mirrored those of the endogenous mouse GLUT4 mRNA. Since previous studies have observed alterations in GLUT4 mRNA levels induced by fasting and refeeding (Sivitz, W. I., DeSautel, S. L., Kayano, T., Bell, G. I., and Pessin, J. E. (1989) Nature 340, 72-74), the regulated expression the hGLUT4[2.4]-CAT transgene was also assessed in these animals. Fasting was observed to decrease CAT activity in white adipose tissue which was super-induced upon refeeding. These alterations in CAT expression occurred in parallel to the changes in endogenous mouse GLUT4 mRNA levels. Although CAT expression in skeletal muscle and brown adipose tissue was unaffected, the endogenous mouse GLUT4 mRNA was also refractory to the effects of fasting/refeeding in these tissues. These data demonstrate that 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene contain all the necessary sequence elements to confer tissue-specific expression and at least some of the sequence elements controlling the hormonal/metabolic regulation of this gene.     

Tissue specific expression of antifreeze protein and growth hormone transgenes driven by the ocean pout (<Emphasis Type="Italic">Macrozoarces americanus</Emphasis>) antifreeze protein OP5a gene promoter in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

Previous research aimed at producing genetically improved salmon broodstock for aquaculture led to the creation of two lines of transgenic Atlantic salmon using gene constructs that were derived in part from the ocean pout OP5a antifreeze protein (AFP) gene. One of the lines was produced using an OP5a AFP gene in which the 5′ region of the promoter was removed (termed t-OP5a-AFP), and the other line contains a growth hormone (GH) transgene (EO-1α) that consists of a chinook salmon GH cDNA driven by a truncated OP5a AFP promoter that is almost identical to that of the t-OP5a-AFP construct. The similarity of the promoter regions of these transgenes provided an opportunity to evaluate their tissue specific expression patterns. Expression of mRNA was evaluated using Northern blot and RT-PCR techniques. The results demonstrate that the AFP and GH trangenes were expressed in almost all body tissues, suggesting that the promoter region of the OP5a AFP gene lacks tissue specific elements. Northern analysis revealed that expression of the t-OP5a-AFP gene was considerably greater than that of the EO-1α GH transgene. Only the spleen tissue of the GH transgenics showed a visible band of hybridization. In contrast clear bands of hybridization were evident in all tissues, except for blood cells, of the AFP transgenics with heart, liver and brain tissue showing the highest levels of mRNA expression. This higher level of expression could be attributable to the presence of introns in the t-OP5a-AFP transgene. Since the GH transgenic salmon grow considerably faster than non-transgenics the low levels of GH transgene expression in this line were clearly sufficient to produce the desired rapid growth phenotype. In contrast the levels of AFP expression were inadequate to impart any improvement in the freeze resistance of the AFP transgenic salmon.     

Enhancer-independent promoter activity of the mouse alphaB-crystallin/small heat shock protein gene in the lens and cornea of transgenic mice

The alphaB-crystallin/small heat shock protein gene is expressed very highly in the mouse eye lens and to a lesser extent in many other nonocular tissues, including the heart, skeletal muscle and brain. Previously we showed in transgenic mice that lens-specific alphaB-crystallin promoter activity is directed by a proximal promoter fragment (-164/+44) and that non-lens promoter activity depends on an upstream enhancer (-427/-259) composed of at least 5 cis-control elements. Here we have used truncated alphaB-crystallin promoter-CAT transgenes to test by biphasic CAT assays and/or histochemistry for specific expression in the cornea and lens. Deletion either of 87 bp (-427/-340) from the 5' end of the alphaB-crystallin enhancer or of the whole enhancer (-427/-258) abolished alphaB-crystallin promoter activity in all tissues except the lens and corneal epithelium when examined by the biphasic CAT assay in 4-5-week-old transgenic mice. These truncations also lowered promoter strength in the lens. The -426/+44-CAT, -339/+44-CAT and -164/+44-CAT (previously thought to be lens-specific in transgenic mice) transgenes were all expressed in the 4-6-week-old corneal epithelium when examined histochemically. Immunohistochemical staining confirmed the presence of endogenous alphaB-crystallin in the mature corneal epithelial cells. CAT gene expression driven by the alphaB-crystallin promoter with or without the enhancer was evident in the embryonic and 4-6-week-old lens. By contrast, activity of the alphaB-crystallin promoter/enhancer-CAT transgene was not detectable in the corneal epithelium before birth. Taken together, these results indicate that the intact enhancer of the alphaB-crystallin/small heat shock protein gene is required for promoter activity in all tissues tested except the lens and cornea.     

Expression of S-antigen in retina, pineal gland, lens, and brain is directed by 5'-flanking sequences.

S-antigen (S-Ag) is an abundant protein of the retina and pineal gland that elicits experimental autoimmune uveitis and pinealocytis in several animal species. To study the elements regulating the expression of S-Ag, we generated transgenic mice expressing the chloramphenicol acetyl transferase (CAT) gene under the control of a 1.3-kilobase pair 5'-flanking segment of the mouse S-Ag gene. While all of the transgenic mice expressed CAT activity in the retina, in some animals CAT activity was also detected in the pineal gland, lens, and brain. Immunoblotting, polymerase chain reaction-mediated detection of RNA, and immunocyto-staining of transgenic tissues with antibodies to CAT and S-Ag established that the profile of expression of the transgene corresponded to that of S-Ag; both proteins were detectable in retinal photoreceptor cells, pinealocytes, lens fiber and epithelial cells, the cerebellum, and the cerebral cortex. These results indicate that S-Ag is expressed in a wider spectrum of the cell types than previously recognized and that a 1.3-kilobase pair S-Ag promoter segment contains sufficient information to direct appropriate tissue-specific gene expression in transgenic mice.     

Osteoblasts are target cells for transformation in c-fos transgenic mice

We have generated transgenic mice expressing the proto-oncogene c-fos from an H-2Kb class I MHC promoter as a tool to identify and isolate cell populations which are sensitive to altered levels of Fos protein. All homozygous H2-c-fosLTR mice develop osteosarcomas with a short latency period. This phenotype is specific for c-fos as transgenic mice expressing the fos- and jun-related genes, fosB and c-jun, from the same regulatory elements do not develop any pathology despite high expression in bone tissues. The c-fos transgene is not expressed during embryogenesis but is expressed after birth in bone tissues before the onset of tumor formation, specifically in putative preosteoblasts, bone- forming osteoblasts, osteocytes, as well as in osteoblastic cells present within the tumors. Primary and clonal cell lines established from c-fos-induced tumors expressed high levels of exogenous c-fos as well as the bone cell marker genes, type I collagen, alkaline phosphatase, and osteopontin/2ar. In contrast, osteocalcin/BGP expression was either low or absent. All cell lines were tumorigenic in vivo, some of which gave rise to osteosarcomas, expressing exogenous c- fos mRNA, and Fos protein in osteoblastic cells. Detailed analysis of one osteogenic cell line, P1, and several P1-derived clonal cell lines indicated that bone-forming osteoblastic cells were transformed by Fos. The regulation of osteocalcin/BGP and alkaline phosphatase gene expression by 1,25-dihydroxyvitamin D3 was abrogated in P1-derived clonal cells, whereas glucocorticoid responsiveness was unaltered. These results suggest that high levels of Fos perturb the normal growth control of osteoblastic cells and exert specific effects on the expression of the osteoblast phenotype.     

Sensitive flow cytometric analysis reveals a novel type of parent-of-origin effect in the mouse genome

The discovery of classic parental imprinting came, at least in part, from the analysis of transgene expression in mice. It was noticed that some transgenes were only expressed following paternal transmission and that others sometimes showed differential patterns of methylation depending on the parent of origin. Here, we present evidence of a novel and more subtle form of parental imprinting by taking advantage of the highly sensitive detection of murine transgene expression afforded by flow cytometry. We have produced nine lines of transgenic mice carrying a GFP reporter linked to the human alpha-globin promoter and enhancer elements, which direct expression to erythroid cells. A high proportion of transgenic lines, four of the nine, display significantly lower levels of expression following maternal transmission. Both the percentage of expressing cells and the mean fluorescence in expressing cells are between 10% and 30% lower following maternal transmission. These effects are reversible upon passage through the opposite germline. This finding raises the possibility that differences in the epigenetic state of the maternal and paternal chromosomes in adult somatic cells are more widespread than was previously thought.