Expression of an Acidothermus cellulolyticus endoglucanase in transgenic rice seeds

The thermostable endo-1,4-β-glucanase (E1) from Acidothermus cellulolyticus, is a useful enzyme for commercial hydrolysis of cellulose into glucose. A codon-optimized synthetic gene encoding this enzyme was transformed into rice (Oryza sativa L. ssp. japonica) under the control of the rice seed storage protein Gt1 promoter. The transgenic line C19 was identified as the one with the highest endoglucanase activity among the total of 36 independent transgenic lines obtained. The cellulase activity in the C19 seeds was estimated at about 830U/g of dried seeds using CMC as substrate. The enzymes produced in the seeds had an optimum pH of 5.0 and optimum temperature of 80°C, which is similar to the enzymes produced by the native bacterium host. This study demonstrates that the transgenic rice seeds could be used as a bioreactor for production of enzymes for cellulosic biomass conversion.     

Expression of a thermostable bacterial cellulase in transgenic tobacco plants

The bacterial gene of the thermostable endo-beta-1,4-glucanase (cellulase) was shown to retain its activity and substrate specificity when expressed in transgenic tobacco plants. The leader peptide of the carrot extensin was efficient in transferring the bacterial enzyme into the apoplast. The expression of the bacterial cellulase gene leads to changes in the plant tissue morphology. In the transgenic plant lines, regeneration of primary shoots from callus occurred at the three to five times higher cytokinin (6-BAP) concentration than in control plants. The transgenic plants that expressed the bacterial gene exhibited increased business and altered leaf shape. The transgenic plants developed can be used as models for studying the cellulases role and function in plants.     

Expression and amplification in transgenic mice of a polyoma virus mutant regulatory region.

Two hybrid gene constructs consisting of wild-type and mutant polyoma regulatory regions fused to a bacterial reporter gene were inserted in the mouse germline. Both transgenes were expressed in a large number of different organs. However, marker gene expression controlled by the polyoma wild-type regulatory region was not detectable in the early embryo and remained low throughout the life of the animal while expression controlled by the polyoma F9-1 mutation was detectable in blastocysts and was significantly higher at later stages of development. The F9-1 hybrid gene was also amplifiable when large T-antigen was supplied in trans to mice or to kidney cells derived from these transgenic mice. Amplification resulted in the appearance of several hundred copies of episomal transgenes and a marked increase of marker gene RNA and protein. Our results suggest that the F9-1 mutation does not alter the target spectrum of gene expression in vivo but does create a more efficient enhancer element in the polyoma early control region. Transgene amplification based upon use of the polyoma regulatory elements may be a means of increasing expression of genes in transgenic mice.     

Expression and characterization of Acidothermus cellulolyticus E1 endoglucanase in transgenic duckweed Lemna minor 8627

Endoglucanase E1 from Acidothermus cellulolyticus was expressed cytosolically under control of the cauliflower mosaic virus 35S promoter in transgenic duckweed, Lemna minor 8627 without any obvious observable phenotypic effects on morphology or rate of growth. The recombinant enzyme co-migrated with the purified catalytic domain fraction of the native E1 protein on western blot analysis, revealing that the cellulose-binding domain was cleaved near or in the linker region. The duckweed-expressed enzyme was biologically active and the expression level was up to 0.24% of total soluble protein. The endoglucanase activity with carboxymethylcellulose averaged 0.2 units mg protein(-1) extracted from fresh duckweed. The optimal temperature and pH for E1 enzyme activity were about 80 degrees C and pH 5, respectively. While extraction with HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) buffer (pH 8) resulted in the highest recovery of total soluble proteins and E1 enzyme, extraction with citrate buffer (pH 4.8) at 65 degrees C enriched relative amounts of E1 enzyme in the extract. This study demonstrates that duckweed may offer new options for the expression of cellulolytic enzymes in transgenic plants.     

Expression of a Clostridium thermocellum endoglucanase gene in Lactobacillus plantarum.

Recombinant plasmid pM25 containing the celE gene of Clostridium thermocellum, which codes for an enzymatically active endoglucanase, was transformed into Lactobacillus plantarum by electroporation. Strains harboring pM25 expressed thermostable endoglucanase, which was found predominantly in the culture medium. Two other plasmids, pGK12 and pSA3, were transformed into L. plantarum, and the stability of each plasmid was evaluated.     

Expression of a dominant negative SHP-2 in transgenic mice induces insulin resistance.

To elucidate the roles of SHP-2, we generated transgenic (Tg) mice expressing a dominant negative mutant lacking protein tyrosine phosphatase domain (DeltaPTP). On examining two lines of Tg mice identified by Southern blot, the transgene product was expressed in skeletal muscle, liver, and adipose tissues, and insulin-induced association of insulin receptor substrate 1 with endogenous SHP-2 was inhibited, confirming that DeltaPTP has a dominant negative property. The intraperitoneal glucose loading test demonstrated an increase in blood glucose levels in Tg mice. Plasma insulin levels in Tg mice after 4 h fasting were 3 times greater with comparable blood glucose levels. To estimate insulin sensitivity by a constant glucose, insulin, and somatostatin infusion, steady state blood glucose levels were higher, suggesting the presence of insulin resistance. Furthermore, we observed the impairment of insulin-stimulated glucose uptake in muscle and adipocytes in the presence of physiological concentrations of insulin. Moreover, tyrosine phosphorylation of insulin receptor substrate-1 and stimulation of phosphatidylinositol 3-kinase and Akt kinase activities by insulin were attenuated in muscle and liver. These results indicate that the inhibition of endogenous SHP-2 function by the overexpression of a dominant negative mutant may lead to impaired insulin sensitivity of glucose metabolism, and thus SHP-2 may function to modulate insulin signaling in target tissues.     

Expression of a hybrid immunoglobulin-T cell receptor protein in transgenic mice

We have constructed a hybrid immunoglobulin (VDJH)-T cell receptor (C alpha) gene using the VDJH exon from a digoxin-specific antibody. This gene was used to make a line of transgenic mice. The hybrid VDJH-C alpha protein is expressed on a subset of T cells in these mice, and we have shown that it forms part of a functional TCR complex by the criteria of coprecipitation and comodulation of CD3 and TCR beta chain components and T cell activation with anti-idiotypic antibodies or digoxin. Furthermore, in cells expressing the hybrid protein, there is allelic exclusion of endogenous TCR alpha genes. We discuss the implications for the comparative structure of T cell receptors and immunoglobulins.     

Expression of CD80 promoter in transgenic mice

CD80 is a very potent co-stimulatory factor which is required for complete T-cell activation. Here, we use transgenic mice as a tool to map the promoter of the CD80 gene. We engineered three different CD80 promoter driven luciferase transgenes: -3084, -1073 and -215. With these transgenes, we have generated three groups of transgenic mice. Our results showed that the -3084 CD80 promoter/luciferase transgene was sufficient to confer tissue-specific expression of the CD80 gene. When the promoter sequence was deleted to -1073, the normal tissue-specific expression was lost. A brain-specific element was mapped between -1073 nt and -215 nt. This element caused up to ninefold higher expression of the CD80 promoter/luciferase in brain tissue of -1073 CD80 promoter/luciferase transgenic animals as compared to -3084 CD80 promoter/luciferase transgenic animals. In contrast to results with a cell culture system, little luciferase activity was detected in -215 CD80 promoter/luciferase transgenic animals.     

Expression of the human angiotensinogen gene in transgenic mice and transfected cells.

We have generated two lines of transgenic mice with integrated copies of a 14-kilobase pair (kb) human DNA fragment containing the angiotensinogen gene, which includes 1.3 kb of 5'- and 3'-flanking regions. In both transgenic lines, a considerable quantity of the correctly initiated and processed angiotensinogen mRNA was detected in the liver and it was detectable in heart. Unexpectedly, mRNA for the transgene was accumulated in the kidney, where is normally the minor source of angiotensinogen, to levels comparable to that in the liver. In addition, an in vitro transfection analysis suggested that the 1.3-kb 5'-flanking sequences are essential for expression of the angiotensinogen gene in hepatic and renal cells and that neither DNA segment within the 14-kb construct contributes significantly to repression of the gene expression in renal cells.     

Human transferrin. Expression and iron modulation of chimeric genes in transgenic mice

Transferrin (TF) is a plasma protein that transports and is regulated by iron. The aim of this study was to characterize human TF gene sequences that respond in vivo to cellular signals affecting expression in various tissues and to iron administration. Chimeric genes were constructed containing 152, 622, and 1152 base pairs (bp) of the human TF5'-flanking region with the coding region of a reporter gene, CAT (chloramphenicol acetyltransferase), and introduced into the germ line of mice. Transgenes containing TF 5'-flanking sequences to -152 bp were expressed poorly in all tissues examined. In contrast, transgenes containing TF sequences to -622 or -1152 bp were expressed at high levels in brain and liver, greater than or equal to 1000-fold higher than tissues such as heart and testes. Liver and brain are major sites of endogenous TF mRNA synthesis, but liver mRNA levels are 10-fold higher than brain. A significant diminution of CAT enzymatic activity in liver accompanied iron administration in both TF(0.67) and TF(1.2)CAT transgenic mice, mimicking the decrease of transferrin in humans following iron overload. Levels of endogenous plasma transferrin also decreased in iron-treated transgenic mice. Transgenic mouse lines carrying human TF chimeric genes will be useful models for analyzing the regulation of human transferrin by iron and for determining the molecular basis of transferrin regulation throughout mammalian development into the aging process.     

Expression of threonine-biosynthetic genes in mammalian cells and transgenic mice

Threonine is a nutritionally essential amino acid (EAA) for the growth and development of humans and other nonruminant animals and must be provided in diets to sustain life. The aim of this study was to synthesize threonine in mammalian cells through transgenic techniques. To achieve this goal, we combined the genes involved in bacterial threonine biosynthesis pathways into a single open reading frame separated by self-cleaving peptides (2A) and then linked it into a transposon system (piggyBac). The plasmids pEF1a-IRES-GFP-E2F-his and pEF1a-IRES-GFP-M2F-his expressed Escherichia coli homoserine kinase and threonine synthase efficiently in mouse cells and enabled cells to synthesize threonine from homoserine. This biosynthetic pathway occurred with a low level of efficiency in transgenic mice. Three transgenic mice were identified by Southern blot from 72 newborn mice, raising the possibility that a high level of expression of these genes in mouse embryos might be lethal. The results indicated that it is feasible to synthesize threonine in animal cells using genetic engineering technology. Further work is required to improve the efficiency of this method for introducing genes into mammals. We propose that the transgenic technology provides a promising means to enhance the synthesis of nutritionally EAAs in farm animals and to eliminate or reduce supplementation of these nutrients in diets for livestock, poultry and fish.     

Expression of retroviral vectors in transgenic mice obtained by embryo infection.

Pre-implantation embryos were infected with the retroviral vector MMCV-neo, which carries the neomycin resistance (neo) gene and the v-myc gene. Three transgenic substrains (M-TKneo 1-3) were derived which stably transmit a single intact copy of the vector. In all of the substrains, expression of the neo gene from the internal thymidine kinase (TK) promoter was detected, with two of the substrains expressing the gene in all tissues analysed. In the third substrain, the vector had integrated on the X chromosome and neo expression varied between different tissues. A second series of transgenic mice were obtained with the retroviral vector SAX, in which the human adenosine deaminase cDNA (ADA) is under the control of an internal SV40 promoter. Four substrains (M-SAX 1-4) were analysed; however, no expression of the ADA cDNA was detected. In all mice, no expression was found of the genes under the control of the viral 5' long terminal repeats (LTRs). In the M-TKneo substrains the vector was hypomethylated irrespective of its expression whereas in the M-SAX mice the vector was hypermethylated. These results demonstrate for the first time that the TK promoter can apparently express a gene in all tissues of adult mice and that retroviral vectors with internal promoters may provide an alternative to DNA injection for the efficient expression of genes in transgenic mice.     

Expression of human lactoferrin in milk of transgenic mice

The expression of human lactoferrin (hLF) in the milk of transgenic mice is described. Regulatory sequences derived from the bovine S1-casein gene were fused to the coding sequence of the hLF cDNA and several lines of transgenic mice were generated. Human LF RNA was detected exclusively in the mammary gland of lactating females and only after the onset of lactation. No aberrant RNA products could be detected using northern blotting and primer extension analysis. The hLF concentrations in the milk ranged from less than 0.1 to 36 g ml^–1. Human LF thus expressed did not differ from human milk derived LF, with respect to molecular mass and immunoreactivity with monoclonal and polyclonal antibodies.     

Expression and regulation of a proenkephalin beta-galactosidase fusion gene in the reproductive system of transgenic mice.

A fusion gene containing 3 kilobases of human proenkephalin 5'-flanking sequences and 1 kilobase of human proenkephalin 3'-flanking sequence and the easily visualized histochemical marker, Escherichia coli beta-galactosidase, was used to study the function of cis-regulatory elements within the human proenkephalin gene in transgenic mice. Here data are presented on expression and regulation of this fusion gene in the reproductive system of three independent lines of transgenic mice. Within the male reproductive system, the fusion gene is expressed in the proximal epididymis and in developing germinal cells but not in mature or elongating spermatids. In the female reproductive system, the transgene was expressed at low basal levels, but expression was dramatically stimulated in the ovary and oviduct by hormonal stimulation and pregnancy; additionally, expression was induced at the uteroplacental junction in pregnant mice. Taken together these observations suggest that critical sequences for expression and regulation of the proenkephalin gene within the reproductive system are contained within sequences of the construct.     

Expression of the chicken transferrin gene in transgenic mice

The chicken transferrin gene was microinjected into the male pronucleus of fertilized mouse eggs, and the eggs were then implanted into foster mothers. Approximately 15%-30% of the offspring from the injected eggs carried chicken DNA sequences; restriction mapping indicated that multiple copies of the chicken gene had integrated into the genome in a tandem arrangement in most of the mice. Six of the seven mice studied expressed the chicken gene, and in five mice there was a 5 to 10 fold preferential expression of chicken transferrin mRNA in liver compared to that in other tissues. Chicken transferrin was secreted into the serum of five of the mice, where it reached steady state concentrations up to 67 micrograms/ml. Offspring from transgenic parents also expressed the chicken gene; in some cases the expression in offspring was very similar to the parent, but in one line expression in offspring had increased 2 to 4 fold.     

Expression of human IFN-gamma genomic DNA in transgenic mice

We have introduced an 8.6-kb fragment of human genomic DNA containing the full length IFN-gamma gene into the mouse germline. The transgenic animals had no biologic or developmental defects as human IFN-gamma does not bind to the mouse IFN R. Regulation of the transgene paralleled that of the endogenous murine IFN-gamma gene in that: 1) it is not expressed constitutively in any tissue examined thus far, 2) it can be induced in thymus and spleen cells by T cell mitogens, 3) it is not expressed in B cells stimulated by LPS, and 4) it produces normal mRNA and biologically active IFN protein. Whereas expression of the transgene is likely restricted to T cells, we had observed that both fibroblasts and B cell lines could express the same DNA when transfected in vitro; this indicates that in vivo, developmental factors restrict expression of the IFN-gamma gene to T cells. These findings also indicate that the 8.6-kb fragment contains the regulatory elements necessary for normal tissue specific expression in vivo. Moreover, they indicate that the regulatory elements for this gene are completely preserved over the phylogenetic distance separating mouse and man, even though substantial drift has occurred in the structural gene, and probably in the IFN-gamma R as well.     

Expression of the hepatitis delta virus large and small antigens in transgenic mice.

Simultaneous infection with hepatitis delta virus (HDV) and hepatitis B virus (HBV) in humans is often associated with severe viral liver disease including fulminant hepatitis. Since HBV is thought to be noncytopathic to the hepatocyte, the enhanced disease severity observed during dual infection has been attributed to either simultaneous immune responses against the two viruses or direct cytotoxic effects of HDV products on the hepatocyte or both. To examine these alternate possibilities, we produced transgenic mice that express the small and large delta antigens (HDAg) in hepatocyte nuclei at levels equal to those observed during natural HDV infection. No biological or histopathological evidence of liver disease was detectable during 18 months of observation, suggesting that neither the large nor small form of HDAg is directly cytopathic to the hepatocyte in vivo.     

Expression of the human insulin gene in transgenic mice

Transgenic mice which specifically express the human insulin gene in the pancreatic beta cells have been obtained. Expression is demonstrated by the presence in the serum of human insulin (determined by a radioimmunoassay for the human C-peptide), the level of which increases upon glucose stimulus, and by the presence of human insulin mRNA in pancreas, but not in the other tissues tested. This result indicates that nucleotide sequences necessary for tissue-specific expression are within the transferred gene and/or in its vicinity. It also suggests that the regulatory molecules are quite similar if not identical in human and mouse.