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PubMed2004

The retinal degeneration (rd) gene seriously impairs spatial cognitive performance in normal and Alzheimer's transgenic mice.

To determine the effects of the recessive retinal degeneration (rd) gene on behavioral performance, three Alzheimer's transgenic lines (APPsw, P301L, APPsw + P301L) and non-transgenic littermates were evaluated in a comprehensive behavioral battery between 5 and 8.5 months of age. For all four genotypes collectively, rd homozygosity resulted in profound impairment in spatial cognitive tasks requiring visual acuity (Morris maze, platform recognition, and radial arm water maze). Non-transgenic and P301L mutant tau mice contributed most to this rd effect since heterozygous and wild type mice performed well. By contrast, spatial cognitive performance of both APPsw-expressing lines was often impaired, irrespective of rd status. Sensorimotor performance was unaffected by rd homozygosity, while rd effects on anxiety were genotype-dependent (less anxiety in NT, APPsw; more anxiety in P301L, APPsw + P301L). Our results strongly encourage rd screening of genetically manipulated mouse lines produced from rd-carrying strain backgrounds to avoid serious potential confounds in the interpretation of spatially based cognitive performance.

Alzheimer Disease↗
PubMed2004

Real-time quantitative PCR-based system for determining transgene copy number in transgenic animals.

In this paper, we describe a rapid and accurate real-time quantitative PCR-based system to determine transgene copy number in transgenic animals. We used the 2(-deltadeltaCt) method to analyze different transgenic lines without the requirement of a control sample previously determined by Southern blot analysis. To determine the transgene copy number in several mouse lines carrying a goat beta-Lactoglobulin transgene, we developed a TaqMan assay in which a goat genomic DNA sample was used as a calibrator. Moreover, we used the glucagon gene as a reference control because this gene is highly conserved between species and amplifies with the same efficiency and sensitivity in goat as in mouse. With this assay, we provide an alternative simple method to determine the transgene copy number, avoiding the traditional and tedious blotting techniques. The assay's discrimination ability from our results is of at least six copies and, similar to the limitations of the blotting techniques, the accuracy of the quantification diminishes when the transgene copy number is high.

Animals↗
PubMed1999

Gene stability in transgenic aspen (Populus). I. Flanking DNA sequences and T-DNA structure.

The stability of transgenes in the genome of transformed plants depends strongly on their correct physical integration into the host genome as well as on flanking target DNA sequences. For long-lived species like trees, however, no information is available so far concerning inactivation or loss of transgenes due to gene silencing or somatic genome rearrangement events. In this study, four independently transformed 35S-rolC transgenic hybrid aspen plants (Populus tremula L. x tremuloides Michx.), each harbouring one copy of the transgene, were investigated during continuous growth in the greenhouse. In one of these transgenic lines (Esch5:35S-rolC-#1) individuals frequently show phenotypic reversions, while in the remaining three lines (Esch5:35S-rolC-#3, -#5, -#16) the gene was essentially stable. Molecular analysis including PCR, Southern and Northern assays clearly showed that the transgene had been lost in the revertant tissue of the unstable line. Sequencing of T-DNA right and left borders, and flanking DNA regions, in all four transgenic aspen lines revealed no differences either in the type of flanking DNA (G-C to A-T ratio) or with respect to the presence of enhancers or MAR (matrix associated repeats)-like structures. Primers located within the left and right flanking regions in the three stable lines could be used to recover the target sites from the untransformed plants. This was not possible, however, with the unstable line, indicating that at least one flanking sequence does not derive from the plant target DNA but is of unknown origin. PCR using other primer pairs, and inverse PCR analysis, revealed an additional truncated T-DNA copy of 1050 nucleotides adjacent to the left border of the complete copy in this line. Sequencing of this truncated T-DNA revealed that it represented an inverted copy of part of the right half of the original construct. This special feature would allow the inverted repeat to pair with right border sequences of the complete copy. This would explain the frequently observed reversion resulting in transgene loss as due to intrachromosomal base-pairing leading to double-stranded loops of single-stranded DNA during mitotic cell divisions.

Bacterial Proteins↗
PubMed1995

SCA1 transgenic mice: a model for neurodegeneration caused by an expanded CAG trinucleotide repeat.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant inherited disorder characterized by degeneration of cerebellar Purkinje cells, spinocerebellar tracts, and selective brainstem neurons owing to the expansion of an unstable CAG trinucleotide repeat. To gain insight into the pathogenesis of the SCA1 mutation and the intergenerational stability of trinucleotide repeats in mice, we have generated transgenic mice expressing the human SCA1 gene with either a normal or an expanded CAG tract. Both transgenes were stable in parent to offspring transmissions. While all six transgenic lines expressing the unexpanded human SCA1 allele had normal Purkinje cells, transgenic animals from five of six lines with the expanded SCA1 allele developed ataxia and Purkinje cell degeneration. These data indicate that expanded CAG repeats expressed in Purkinje cells are sufficient to produce degeneration and ataxia and demonstrate that a mouse model can be established for neurodegeneration caused by CAG repeat expansions.

Animals↗
PubMed2006

Pyrolysis-field ionization mass spectrometry of rhizodeposits - a new approach to identify potential effects of genetically modified plants on soil organisms.

The objectives of the present study were (1) to investigate the qualitative composition of rhizodeposits leached from soils cropped with non-transgenic and genetically modified (GM) potatoes, and disclose if there were GM-specific modifications in potato rhizodeposition, and (2) to compare these results with conventional bulk parameters of microbial activity in soil. We have raised potatoes from a non-transgenic line (Solanum tuberosum L. cv. Désirée) and three GM lines, which expressed a gene for the resistance to kanamycin (DLH 9000) and a gene for T4 lysozyme (DL10 and DL12). A sandy soil placed in 340 cm3-"CombiSart" containers was used, from which the rhizodeposit was leached after a six-week growth period. The freeze-dried leachates were analyzed by pyrolysis-field ionization mass spectrometry (Py-FIMS). The Py-FI mass spectra gave detailed molecular-chemical information about the composition of leachates, indicating that the potato growth generally altered the composition of the soil solution. Moreover, a principal component analysis of the mass spectra showed differences between the leachates from the non-transgenic parent line and the GM potatoes as well as among the latter group. However, these differences in molecular composition could not be assigned to the release of T4-lysozyme into soil. Dehydrogenase activity and substrate-induced soil respiration as more common bulk parameters of soil microbial activity failed to disclose any significant effects of the various potatoes grown. The limitations of the described rhizodeposit leaching and analysis for risk assessment of GM potato cropping under field conditions are discussed critically. However, it could be concluded that the Py-FI mass spectrometric "fingerprint" can be developed as a fast, comprehensive, highly sensitive and reproducible analytical approach to discern any effects GM-crops may exert on soil ecological parameters.

Animals↗
PubMed2006

Health status of transgenic pigs expressing the human complement regulatory protein CD59.

BACKGROUND: Microinjection of foreign DNA into pronuclei of zygotes has been the method of choice for the production of transgenic domestic animals. Following microinjection the transgene is randomly integrated into the host genome which can be associated with insertional mutagenesis and unwanted pathological side effects. METHODS: Here, we evaluated the health status of pigs transgenic for the human regulator of complement activation (RCA) CD59 and conducted a complete pathomorphological examination on 19 RCA transgenic pigs at 1 to 32 months of age from nine transgenic lines. Nine wild-type animals served as controls. Expression levels of human complement regulator CD59 (hCD59) mRNA were measured by RT-PCR and distribution of hCD59 protein was determined by immunohistochemistry. RESULTS: Albeit variable transgene expression levels, no specific pathomorphologic phenotype associated with the presence of the transgene in all analyzed pig lines could be detected. CONCLUSIONS: Transgenic expression of this human RCA gene construct is not correlated with a specific pathological phenotype in pigs. This is crucial for the application of the technology and the use of transgenic pigs for biomedical and agricultural applications.

Animals↗
PubMed2003

The sweet pepper ferredoxin-like protein (pflp) conferred resistance against soft rot disease in Oncidium orchid.

Genetic engineering to date has not been used to introduce disease resistance genes into the orchid gene pool. The ferredoxin-like protein gene originally isolated from sweet pepper is thought to function as a natural defense against infection due to its antimicrobial properties. Hence it was reasoned that introduction of this gene might produce Oncidium plants resistant to Erwinia carotovora, the causal agent for the soft rot disease. An expression vector containing sweet pepper ferredoxin-like protein (pflp) cDNA, hph and gusA coding sequence was successfully transformed into protocorm-like bodies (PLBs) of Oncidium orchid, using Agrobacterium tumefaciens strain EHA105. A total of 17 independent transgenic orchid lines was obtained, out of which six transgenic lines (beta-glucuronidase (GUS) positive) were randomly selected and confirmed by Southern, northern and western blot analyses. A bioassay was conducted on the transgenic lines. Transgenic plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen. Our results suggest that pflp may be an extremely useful gene for genetic engineering strategies in orchids to confer resistance against soft rot disease.

Capsicum↗
PubMed2005

High expression of foot-and-mouth disease virus structural protein VP1 in tobacco chloroplasts.

A tobacco chloroplast expression vector, pTRVP1, containing the foot-and-mouth disease virus (FMDV) VP1 gene and the selective marker aadA gene, was constructed and transferred to tobacco by biolistic method. Three resistant lines were obtained through spectinomycin selection, and each transgenic line was subjected to a second round of spectinomycin selection. PCR and PCR southern blot analysis revealed that the VP1 gene had integrated into the chloroplast genome. Western blot and quantification ELISA assays indicated that the VP1 gene was expressed in tobacco chloroplasts and accounted for 2-3% of total soluble protein. This suggested that plant chloroplasts were an efficient expression system for the potential production of recombinant antigens in plants.

Capsid Proteins↗
PubMed2006

Expression of the lipid transfer protein Ace-AMP1 in transgenic wheat enhances antifungal activity and defense responses.

To enhance fungal disease resistance, wheat plants (cv. Bobwhite) were engineered to constitutively express the potent antimicrobial protein Ace-AMP1 from Allium cepa, driven by a maize ubiquitin promoter along with its first intron. The bar gene was used for selection of putative transformants on medium containing phosphinothricin (PPT). Transgene inheritance, integration and stability of expression were confirmed over two generations by PCR, Southern, northern and western blot analyses, respectively. The levels of Ace-AMP1 in different transgenic lines correlated with the transcript levels of the transgene. Up to 50% increase in resistance to Blumeria graminis f. sp. tritici was detected in detached leaf assays. In ears of transgenic wheat inoculated with Neovossia indica, Ace-AMP1 intensified expression of defense-related genes. Elevated levels of salicylic acid and of transcripts of phenylalanine ammonia lyase (PAL), glucanase (PR2) and chitinase (PR3) in the transgenic plants indicated manifestation of systemic acquired resistance (SAR).

Antifungal Agents↗
PubMed1998

Overexpression of a minor component of myelin basic protein isoform (17.2 kDa) can restore myelinogenesis in transgenic shiverer mice.

Shiverer (shi) mice, which are neurologically mutant, lack a large portion of the gene for the myelin basic proteins (MBPs), have virtually no myelin in their central nervous system (CNS), and shiver, undergo seizures, and die early. At least five types of MBPs (21.5, 18.5, 17.3, 17.2 and 14.0 kDa) are known to be generated through alternative splicing from a single MBP gene. We have produced transgenic shi mice carrying a cDNA encoding mouse 14-kDa MBP isoform, the most abundant form of MBPs, under control of a mouse MBP gene promoter, and showed that expression of the 14-kDa MBP can restore CNS myelination. To test whether the 17.2-kDa MBP isoform, one of the minor components of MBPs, can also elicit myelination in homozygous shi mutants, we produced seven independent transgenic shi mice carrying cDNA encoding the mouse 17.2-kDa MBP isoform, and the transcription of which was driven by a mouse MBP gene promoter. The axons in the cerebellum of one transgenic line, which exhibited the highest expression of transgene-derived mRNA ( approximately 50% of the level of total MBP mRNA in the normal mouse brain), were myelinated. This mouse exhibited nearly normal behavior. These findings indicate that the 17.2-kDa MBP isoform, even when the only 17.2-kDa MBP isoform is present, has the ability to elicit CNS myelination in transgenic shi mice. This transgenic strategy will be useful for elucidating the role of each type of MBP isoform in CNS myelinogenesis.

Alternative Splicing↗
PubMed2003

Renal tubule-specific expression and urinary secretion of human growth hormone: a kidney-based transgenic bioreactor growth.

Tissue-specific expression of human genes and secretion of human proteins into the body fluids in transgenic animals provides an important means of manufacturing large-quantity and high-quality pharmaceuticals. The present study demonstrates using transgenic mice that a 3.0 kb promoter of the mouse Tamm-Horsfall protein (THP, or uromodulin) gene directs the specific expression of human growth hormone (hGH) gene in the kidney followed by the secretion of hGH protein into the urine. hGH expression was detected in renal tubules that actively produce the THP, that is, the ascending limb of Henle's loop and distal convoluted tubules. Up to 500 ng/ml of hGH was detected in the urine, and this level remained constant throughout the 10-month observation period. hGH was also detectable in the stomach epithelium and serum in two of the transgenic lines, suggesting position-dependent effects of the transgene and leakage of hGH from the site of synthesis into the bloodstream, respectively. These results indicate that the 3.0 kb mouse THP promoter is primarily kidney-specific and can be used to convert kidney into a bioreactor in transgenic animals to produce recombinant proteins. Given the capacity of urine production independent of age, sex and lactation, the ease of urinary protein purification, and the potentially distinct machinery for post-translational modifications in the kidney epithelial cells, the kidney-based transgenic bioreactor may offer unique opportunities for producing certain complex pharmaceuticals.

Animals↗
PubMed1993

Juvenile cystic kidneys (jck): a new mouse mutation which causes polycystic kidneys.

We have characterized a new recessive mutation in the mouse which predisposes to the development of polycystic kidney disease. This mutation, called juvenile cystic kidneys (jck), arose in a transgenic line of mice, but appears unrelated to the transgene since it segregates freely from it. While focal cysts are evident in affected animals as early as three days of life and the disease is progressive, the mice are fertile and generally survive to four or more months of age. Complementation analysis indicates that the jck mutation is not allelic with three other known recessive polycystic kidney mutations (cpk and two as yet unnamed mutations), and linkage studies demonstrate it is unlikely to be allelic with a fourth (pcy). The study of these five mutations and their interactions should prove useful for understanding the mechanisms required to maintain the normal integrity of renal tubules.

Alleles↗
PubMed2003

Investigating the regulation of one-carbon metabolism in Arabidopsis thaliana.

Serine (Ser) biosynthesis in C(3) plants can occur via several pathways. One major route involves the tetrahydrofolate (THF)-dependent activities of the glycine decarboxylase complex (GDC, EC 2.1.1.10) and serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) with glycine (Gly) as one-carbon (1-C) source. An alternative THF-dependent pathway involves the C1-THF synthase/SHMT activities with formate as 1-C source. Here, we have investigated aspects of the regulation of these two folate-mediated pathways in Arabidopsis thaliana (L.) Heynh. Columbia using two approaches. Firstly, transgenic plants overexpressing formate dehydrogenase (FDH, EC 1.2.1.2) were used to continue our previous studies on the function of FDH in formate metabolism. The formate pool size was approximately 73 nmol (g FW)(-1) in wild type (WT) Arabidopsis plants; three independent transgenic lines had similar-sized pools of formate. Transgenic plants produced more (13)CO(2) from supplied [(13)C]formate than did WT plants but were not significantly different from WT plants in their synthesis of Ser. We concluded that FDH has no direct role in the regulation of the above two pathways of Ser synthesis; the breakdown of formate to CO(2) by the FDH reaction is the primary and preferred fate of the organic acid in Arabidopsis. The ratio between the GDC/SHMT and C1-THF synthase/SHMT pathways of Ser synthesis from [alpha-(13)C]Gly and [(13)C]formate, respectively, in Arabidopsis shoots was 21 : 1; in roots, 9 : 1. In shoots, therefore, the pathway from formate plays only a small role in Ser synthesis; in the case of roots, results indicated that the 9 : 1 ratio was as a result of greater fluxes of (13)C through both pathways together with a relatively higher contribution from the C1-THF synthase/SHMT route than in shoots. We also examined the synthesis of Ser in a GDC-deficient mutant of Arabidopsis (glyD) where the GDC/SHMT pathway was impaired. Compared with WT, glyD plants accumulated 5-fold more Gly than WT after supplying [alpha-(13)C]Gly for 24 h; the accumulation of Ser from [alpha-(13)C]Gly was reduced by 25% in the same time period. On the other hand, the accumulation of Ser through the C1-THF synthase/SHMT pathway in glyD plants was 2.5-fold greater than that in WT plants. Our experiments confirmed that the GDC/SHMT and C1-THF synthase/SHMT pathways normally operate independently in Arabidopsis plants but that when the primary GDC/SHMT pathway is impaired the alternative C1-THF synthase/SHMT pathway can partially compensate for deficiencies in the synthesis of Ser.

Aminohydrolases↗
PubMed2002

Tyr-TGFalpha transgenic mice develop ocular melanocytic lesions.

Transforming growth factor-alpha (TGFalpha) has been implicated in melanocyte transformation, as it is expressed in melanocytic lesions and in melanoma cells. We investigated its role in melanoma development using a transgenic mouse model. The mice were generated by microinjection of a transgene with 270 bp of the mouse tyrosinase promoter and the cDNA for human TGFalpha. No significant skin abnormalities were found, but individuals from three transgenic lines developed ocular melanocytoses (seven out of 10 transgenics), usually after a long latency period. In particular, the melanocyte component of the choroid was thicker than in non-transgenic controls, consistent with hyperplasia. The retinal pigment epithelium was unaffected. Melanocytic lesions were also present in the posterior eye, and abnormal distributions of melanocytes were found in neural tissue of the brain, skeletal muscle of the head and the Harderian glands, indicating migration from the choroid. It was concluded that mice engineered to express the normal growth factor TGFalpha from a tyrosinase promoter spontaneously developed melanocytic lesions in the eye but not the skin.

Animals↗
PubMed2003

RPS4-mediated disease resistance requires the combined presence of RPS4 transcripts with full-length and truncated open reading frames.

Arabidopsis RPS4 belongs to the Toll/interleukin-1 receptor (TIR)-nucleotide binding site (NBS)-Leu-rich repeat (LRR) class of disease resistance (R) genes. Like other family members in different plant species, RPS4 produces alternative transcripts with truncated open reading frames. The dominant alternative RPS4 transcripts are generated by retention of intron 3 or introns 2 and 3, which contain in-frame stop codons and lie downstream of the NBS-encoding exon. We analyzed the biological significance of these alternative transcripts in disease resistance by removing introns 2 and 3, either individually or in combination, from a functional RPS4-Ler (Landsberg erecta) transgene. Removal of one or both introns abolished the function of the RPS4 transgene, whereas expression was not affected. In addition, a truncated RPS4-Ler transgene encoding the putative TIR and NBS domains was not sufficient to confer resistance, suggesting that the combined presence of regular and alternative RPS4 transcripts is necessary for function. Interestingly, we observed partial resistance in transgenic lines expressing both intron-deficient and truncated transgenes. This finding confirms the requirement for regular and alternative RPS4 transcripts and indicates that alternative transcripts function at the protein level rather than as regulatory RNAs. Together with published results on the tobacco N gene, our data suggest that the generation of alternative TIR-NBS-LRR R gene transcripts is of general biological significance across plant species.

Arabidopsis↗
PubMed2003

Adeno-associated virus type 2-mediated gene transfer: role of cellular T-cell protein tyrosine phosphatase in transgene expression in established cell lines in vitro and transgenic mice in vivo.

The use of adeno-associated virus type 2 (AAV) vectors has gained attention as a potentially useful alternative to the more commonly used retrovirus and adenovirus vectors for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein (FKBP52), interacts with the single-stranded D sequence within the AAV inverted terminal repeats, inhibits viral second-strand DNA synthesis, and consequently limits high-efficiency transgene expression (K. Qing, J. Hansen, K. A. Weigel-Kelley, M. Tan, S. Zhou, and A. Srivastava, J. Virol., 75: 8968-8976, 2001). FKBP52 can be phosphorylated at both tyrosine and serine/threonine residues, but only the phosphorylated forms of FKBP52 interact with the D sequence. Furthermore, the tyrosine-phosphorylated FKBP52 inhibits AAV second-strand DNA synthesis by greater than 90%, and the serine/threonine-phosphorylated FKBP52 causes approximately 40% inhibition, whereas the dephosphorylated FKBP52 has no effect on AAV second-strand DNA synthesis. In the present study, we have identified that the tyrosine-phosphorylated form of FKBP52 is a substrate for the cellular T-cell protein tyrosine phosphatase (TC-PTP). Deliberate overexpression of the murine wild-type (wt) TC-PTP gene, but not that of a cysteine-to-serine (C-S) mutant, caused tyrosine dephosphorylation of FKBP52, leading to efficient viral second-strand DNA synthesis and resulting in a significant increase in AAV-mediated transduction efficiency in HeLa cells in vitro. Both wt and C-S mutant TC-PTP expression cassettes were also used to generate transgenic mice. Primitive hematopoietic stem/progenitor cells from wt TC-PTP-transgenic mice, but not from C-S mutant TC-PTP-transgenic mice, could be successfully transduced by recombinant AAV vectors. These studies corroborate the fact that tyrosine phosphorylation of the cellular FKBP52 protein strongly influences AAV transduction efficiency, which may have important implications in the optimal use of AAV vectors in human gene therapy.

Animals↗
PubMed1996

Beta-MHC and SMLC1 transgene induction in overloaded skeletal muscle of transgenic mice.

The hypertrophic responses of white fast-twitch muscle to mechanical overload has been investigated using transgenic mice. After 7 wk of overload, endogenous beta-myosin heavy chain (MHC) and slow myosin light chain 1 and 2 (SMLC1, SMLC2) protein were increased in the overloaded plantaris (OP) muscle compared with sham-operated control plantaris (CP)muscle. Concurrently, the levels of endogenous beta-MHC, SMLC1, SMLC2, and cardiac/slow troponin C (CTnC) mRNA transcripts were significantly increased in OP muscles, whereas skeletal troponin C (sTnC) mRNA transcript levels decreased. As an initial attempt to locate DNA sequence(s) that governs beta-MHC induction in response to mechanical overload, multiple independent transgenic lines harboring four different human beta-MHC transgenes (beta 1286, beta 988, beta 450, beta 141) were generated. Except for transgene beta 141, muscle-specific expression and induction (3- to 22-fold) in OP muscles were observed by measuring chloramphenicol acetyltransferase activity (CAT assay). Induction of a SMLC1 transgene (3920SMLC1) in OP muscles was also observed. Collectively, these in vivo data provide evidence that 1) a mechanical overload inducible element(s) is located between nucleotides -450 and +120 of the human beta-MHC transgene, 2) 3,900 bp of 5' sequence is sufficient to confer mechanical overload induction of a SMLC1 transgene, and 3) the increased expression of slow/type I isomyosin (beta-MHC, SMLC1, SMLC2) in response to mechanical overload is regulated, in part, transcriptionally.

Animals↗