Hormonal and nutritional regulation of muscle carnitine palmitoyltransferase I gene expression in vivo

Transgenic mice carrying the human heart muscle carnitine palmitoyltransferase I (M-CPTI) gene fused to a CAT reporter gene were generated to study the regulation of M-CPTI gene expression. When the mice were fasted for 48 h, CAT activity and mRNA levels increased by more than 2-fold in heart and skeletal muscle, but not liver or kidney. In the diabetic transgenic mice, there was a 2- to 3-fold increase in CAT activity and CAT mRNA levels in heart and skeletal muscle which upon insulin administration reverted to that observed with the control insulin sufficient transgenic mice. Feeding a high fat diet increased CAT activity and mRNA levels by 2- to 4-fold in heart and skeletal muscle of the transgenic mice compared to the control transgenic mice on regular diet. Overall, the M-CPTI promoter was found to be necessary for the tissue-specific hormonal and dietary regulation of the gene expression.     

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.     

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.     

Production of transgenic quails with high frequency of germ-line transmission using VSV-G pseudotyped retroviral vector

We report here the production of transgenic quails using a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus (MoMLV) pseudotyped with vesicular stomatitis virus G protein (VSV-G). The retroviral vector was injected into laid quail embryos at the blastodermal stage, and the embryos were incubated to hatch to produce G(0) transgenic quails. Among 134 embryos subjected to viral injection, 37 hatched. The viral vector sequence was detected in the tissues of all G(0) quails. The germ-line transmission efficiency of G(0) quails mated with nontransgenic quails was more than 80% on average. Southern blot analysis revealed that the G(1) transgenic progeny had one to three copies of the transgene. The expression of vector-encoded neomycin-resistance gene under the control of the Rous sarcoma virus (RSV) promoter was observed in several tissues including heart and muscle of both G(1) and G(2) transgenic offspring. Due to the high frequency of germ-line transmission, this method may markedly facilitate the production of transgenic avian.     

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole‐mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker‐mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2‐kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5‐kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8‐kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters. Dev. Genet. 25:158–167, 1999. © 1999 Wiley‐Liss, Inc.     

Factors affecting the use of chloramphenicol acetyltransferase as a marker for Brassica genetic transformation

The CAT gene which codes for the enzyme chloramphenicol acetyltransferase was found to be ineffective as a reporter gene in cells and tissues of Brassica species. High levels of endogenous CAT activity were found to be widespread among this genus and did not appear to be distributed in a tissue- or cell-specific manner. Moreover, the presence of an inhibitor of CAT activity was discovered in Brassica napus and Brassica juncea. This inhibitor appeared to act selectively on bacterial CAT in transgenic plants. These findings provided an explanation for difficulties experienced in the detection of transgenic CAT activity in B. napus.     

Two 5'-regions are required for nutritional and insulin regulation of the fatty-acid synthase promoter in transgenic mice

We previously reported that 2.1 kilobase pairs of the 5'-flanking sequence are sufficient for tissue-specific and hormonal/metabolic regulation of the fatty-acid synthase (FAS) gene in transgenic mice. We also demonstrated that the -65 E-box is required for insulin regulation of the FAS promoter using 3T3-L1 adipocytes in culture. To further define sequences required for FAS gene expression, we generated transgenic mice carrying from -644, -444, -278, and -131 to +67 base pairs of the rat FAS 5'-flanking sequence fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Similar to the expression observed with -2100-FAS-CAT transgenic mice, transgenic mice harboring -644-FAS-CAT and -444-FAS-CAT expressed high levels of CAT mRNA only in lipogenic tissues (liver and adipose tissue) in a manner identical to the endogenous FAS mRNA. In contrast, -278-FAS-CAT and -131-FAS-CAT transgenic mice did not show appreciable CAT expression in any of the tissues examined. When previously fasted mice were refed a high carbohydrate, fat-free diet, CAT mRNA expression in transgenic mice harboring -644-FAS-CAT and -444-FAS-CAT was induced dramatically in liver and adipose tissue. The induction was virtually identical to that observed in -2100-FAS-CAT transgenic mice and to the endogenous FAS mRNA. In contrast, -278-FAS-CAT transgenic mice showed induction by feeding, but at a much lower magnitude in both liver and adipose tissue. The -131-FAS-CAT transgenic mice did not show any CAT expression either when fasted or refed a high carbohydrate diet. To study further the effect of insulin, we made these transgenic mice insulin-deficient by streptozotocin treatment. Insulin administration to the streptozotocin-diabetic mice increased CAT mRNA levels driven by the -644 FAS and -444 FAS promoters in liver and adipose tissue, paralleling the endogenous FAS mRNA levels. In the case of -278-FAS-CAT, the induction observed was at a much lower magnitude, and deletion to -131 base pairs did not show any increase in CAT expression by insulin. This study demonstrates that the sequence requirement for FAS gene regulation employing an in vitro culture system does not reflect the in vivo situation and that two 5'-flanking regions are required for proper nutritional and insulin regulation of the FAS gene. Cotransfection of the upstream stimulatory factor and various FAS promoter-luciferase constructs as well as in vitro binding studies suggest a function for the upstream stimulatory factor at both the -65 and -332 E-box sequences.     

Robust and ubiquitous GFP expression in a single generation of chicken embryos using the avian retroviral vector,RCASBP

Functional genomics in avian models has lagged behind that of mammals, and the production of transgenic birds has proven to be challenging and time-consuming. All current methods rely upon breeding chimeric birds through at least one generation. Here, we report a rapid method for the ubiquitous expression of GFP in chicken embryos in a single generation (G-0), using the avian retroviral vector, Replication-Competent Avian sarcoma-leukosis virus, with a Splice acceptor, Bryan RSV Pol (RCASBP). High-titre RCASBP retrovirus carrying eGFP was injected into unincubated (stage X) blastoderms in ovo. This resulted in stable and widespread expression of eGFP throughout development in a very high proportion of embryos. Transgenic tissues were identified by fluorescence and immunohistochemistry. These results indicate that chicken blastodermal cells are permissive for infection by the RCASBP virus. This system represents a rapid and efficient method of producing global gene expression in the chicken embryo. The method can be used to generate avian cells with a stable genetic marker, or to induce global expression of a gene of choice. Interestingly, in day 8.5 embryos, somatic cells the embryonic gonads were predominantly GFP positive but primordial germ cells were GFP negative, indicating viral silencing in the embryonic germline. This dichotomy in the gonads allows the isolation or enrichment of the germ cells through negative selection during embryonic stages. This transgenic chicken model is of value in developmental studies, and for the isolation and study of avian primordial germ cells.     

Localization of 9E3/CEF-4 in avian tissues: expression is absent in Rous sarcoma virus-induced tumors but is stimulated by injury

The avian gene 9E3/CEF-4, a member of the superfamily of genes that includes KC and gro, is expressed abundantly in exponentially growing cultures of chick embryo fibroblasts (CEFs) and at high levels in CEFs transformed with Rous sarcoma virus (RSV). The product of this gene is a secreted protein that has homologies and structural similarities to inflammatory mediators. The function of 9E3 is obscure and its expression in vivo has not yet been investigated. We studied by in situ hybridization and RNA blots the pattern of 9E3 mRNA distribution in the wings of normal, wounded, and RSV-infected newly hatched chicks. We found that the message for 9E3 is high in specific tissues in normal wings; whereas connective tissue, tendon, and bone express the gene, muscle fibers, endothelium, epidermis, and bone marrow do not. The distribution coincides with that of interstitial collagen. Wounding results in marked elevation of the mRNA within the granulation tissue formed during healing and in adjacent tissues, especially those showing neovascularization. Similar elevation of mRNA occurs immediately adjacent to RSV tumors but, surprisingly, the tumor tissue itself shows no detectable levels of this message. Cells explanted from the tumors and grown in culture also show no expression of 9E3, in marked contrast to the very high level found in similarly cultured RSV-transformed CEFs. These results show that there are intrinsic differences between transformed embryonic cells in tissue culture and RSV target cells in the hatched chick. However, the expression of the gene in the periphery of tumors leaves open the possibility that 9E3 may still be involved in RSV carcinogenesis. The abundant expression of 9E3 in normal tissues indicates that the product of this gene plays a normal physiological role in tissues growing by cell division, perhaps as a growth regulator. The elevated expression of 9E3 in areas of neovascularization, makes it possible that the product of this gene could act as an angiogenic factor. Finally, expression in conjunction with high collagen levels and in wounded tissues may point to a role in wound response and/or repair, possibly via alteration of extracellular matrix.     

Rous sarcoma virus does not induce sarcoma in early chick embryos by lack of the v-src gene expression.

The Schmidt-Ruppin or the B77 strain of Rous sarcoma virus (RSV) was inoculated into limb buds of 4.5-days-old avian embryos. No sarcoma but blister formation was observed in those RSV-inoculated embryos. Protein kinase activity of pp60v-src in RSV-inoculated embryos, even in the site of virus inoculation, was the same as that in mock-infected embryos. This indicated that the expression of the v-src gene did not attain superiority over that of the c-src gene in RSV-inoculated embryos. The v-src gene was detected in every DNA from tissues of RSV-inoculated embryos but not in DNAs from tissues of RSV-inoculated chicken except for the DNA from Rous sarcoma. Those results confirmed that the lack of sarcoma induction in early avian embryos by RSV was due to the lack of the expression of the v-src gene which was present in the target cells.     

A compact unc45b‐promoter drives muscle‐specific expression in zebrafish and mouse

Summary: Gene therapeutic approaches to cure genetic diseases require tools to express the rescuing gene exclusively within the affected tissues. Viruses are often chosen as gene transfer vehicles but they have limited capacity for genetic information to be carried and transduced. In addition, to avoid off‐target effects the therapeutic gene should be driven by a tissue‐specific promoter in order to ensure expression in the target organs, tissues, or cell populations. The larger the promoter, the less space will be left for the respective gene. Thus, there is a need for small but tissue‐specific promoters. Here, we describe a compact unc45b promoter fragment of 195 bp that retains the ability to drive gene expression exclusively in skeletal and cardiac muscle in zebrafish and mouse. Remarkably, the described unc45b promoter fragment not only drives muscle‐specific expression but presents heat‐shock inducibility, allowing a temporal and spatial quantity control of (trans)gene expression. Here, we demonstrate that the transgenic expression of the smyd1b gene driven by the unc45b promoter fragment is able to rescue the embryonically lethal heart and skeletal muscle defects in smyd1b‐deficient flatline mutant zebrafish. Our findings demonstrate that the described muscle‐specific unc45b promoter fragment might be a valuable tool for the development of genetic therapies in patients suffering from myopathies. genesis 54:431–438, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Functional variability of the Lr34 durable resistance gene in transgenic wheat

Breeding for durable disease resistance is challenging, yet essential to improve crops for sustainable agriculture. The wheat Lr34 gene is one of the few cloned, durable resistance genes in plants. It encodes an ATP binding cassette transporter and has been a source of resistance against biotrophic pathogens, such as leaf rust (Puccinina triticina), for over 100 years. As endogenous Lr34 confers quantitative resistance, we wanted to determine the effects of transgenic Lr34 with specific reference to how expression levels affect resistance. Transgenic Lr34 wheat lines were made in two different, susceptible genetic backgrounds. We found that the introduction of the Lr34 resistance allele was sufficient to provide comparable levels of leaf rust resistance as the endogenous Lr34 gene. As with the endogenous gene, we observed resistance in seedlings after cold treatment and in flag leaves of adult plants, as well as Lr34‐associated leaf tip necrosis. The transgene‐based Lr34 resistance did not involve a hypersensitive response, altered callose deposition or up‐regulation of PR genes. Higher expression levels compared to endogenous Lr34 were observed in the transgenic lines both at seedling as well as adult stage and some improvement of resistance was seen in the flag leaf. Interestingly, in one genetic background the transgenic Lr34‐based resistance resulted in improved seedling resistance without cold treatment. These data indicate that functional variability in Lr34‐based resistance can be created using a transgenic approach.