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1.  Strict control of transgene expression in a mouse model for sensitive biological applications based on RMCE compatible ES cells 
Nucleic Acids Research  2010;39(1):e1.
Recombinant mouse strains that harbor tightly controlled transgene expression proved to be indispensible tools to elucidate gene function. Different strategies have been employed to achieve controlled induction of the transgene. However, many models are accompanied by a considerable level of basal expression in the non-induced state. Thereby, applications that request tight control of transgene expression, such as the expression of toxic genes and the investigation of immune response to neo antigens are excluded. We developed a new Cre/loxP-based strategy to achieve strict control of transgene expression. This strategy was combined with RMCE (recombinase mediated cassette exchange) that facilitates the targeting of genes into a tagged site in ES cells. The tightness of regulation was confirmed using luciferase as a reporter. The transgene was induced upon breeding these mice to effector animals harboring either the ubiquitous (ROSA26) or liver-specific (Albumin) expression of CreERT2, and subsequent feeding with Tamoxifen. Making use of RMCE, luciferase was replaced by Ovalbumin antigen. Mice generated from these ES cells were mated with mice expressing liver-specific CreERT2. The transgenic mice were examined for the establishment of an immune response. They were fully competent to establish an immune response upon hepatocyte specific OVA antigen expression as indicated by a massive liver damage upon Tamoxifen treatment and did not show OVA tolerance. Together, this proves that this strategy supports strict control of transgenes that is even compatible with highly sensitive biological readouts.
PMCID: PMC3017619  PMID: 20935052
2.  Toxin–antitoxin based transgene expression in mammalian cells 
Nucleic Acids Research  2009;38(5):e32.
Long-term, recombinant gene expression in mammalian cells depends on the nature of the transgene integration site and its inherent properties to modulate transcription (epigenetic effects). Here we describe a method by which high transgene expression is achieved and stabilized in extensively proliferating cultures. The method is based on strict co-expression of the transgene with an antitoxin in cells that express the respective toxin. Since the strength of antitoxin expression correlates with an advantage for cell growth, the cells with strong antitoxin expression are enriched over time in cultures of heterogeneous cells. This principle was applied to CHO cell lines that conditionally express the toxin kid and that are transduced to co-express the antitoxin kis together with different transgenes of interest. Cultivation of pools of transfectants that express the toxin steadily increase their transgene expression within several weeks to reach a maximum that is up to 120-fold over the initial status. In contrast, average transgene expression drops in the absence of toxin expression. Together, we show that cells conditionally expressing kid can be employed to create overexpressing cells by a simple coupling of kis to the transgene of interest, without further manipulation and in absence of selectable drugs.
PMCID: PMC2836568  PMID: 20007149
3.  The genome of the simian and human malaria parasite Plasmodium knowlesi 
Nature  2008;455(7214):799-803.
Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the ‘kra’ monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia1,2. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated3, and it has a close phylogenetic relationship to Plasmodium vivax​4, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or ‘hypnozoite’ in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone5) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome4 and other sequenced Plasmodium genomes6-8. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs9, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.
PMCID: PMC2656934  PMID: 18843368
4.  Subclinical pulmonary vein narrowing after ablation for atrial fibrillation 
Heart  2005;91(5):672-673.
PMCID: PMC1768907  PMID: 15831661
atrial fibrillation; catheter ablation; magnetic resonance; pulmonary vein
5.  The genome of the social amoeba Dictyostelium discoideum 
Eichinger, L. | Pachebat, J.A. | Glöckner, G. | Rajandream, M.-A. | Sucgang, R. | Berriman, M. | Song, J. | Olsen, R. | Szafranski, K. | Xu, Q. | Tunggal, B. | Kummerfeld, S. | Madera, M. | Konfortov, B. A. | Rivero, F. | Bankier, A. T. | Lehmann, R. | Hamlin, N. | Davies, R. | Gaudet, P. | Fey, P. | Pilcher, K. | Chen, G. | Saunders, D. | Sodergren, E. | Davis, P. | Kerhornou, A. | Nie, X. | Hall, N. | Anjard, C. | Hemphill, L. | Bason, N. | Farbrother, P. | Desany, B. | Just, E. | Morio, T. | Rost, R. | Churcher, C. | Cooper, J. | Haydock, S. | van Driessche, N. | Cronin, A. | Goodhead, I. | Muzny, D. | Mourier, T. | Pain, A. | Lu, M. | Harper, D. | Lindsay, R. | Hauser, H. | James, K. | Quiles, M. | Babu, M. Madan | Saito, T. | Buchrieser, C. | Wardroper, A. | Felder, M. | Thangavelu, M. | Johnson, D. | Knights, A. | Loulseged, H. | Mungall, K. | Oliver, K. | Price, C. | Quail, M.A. | Urushihara, H. | Hernandez, J. | Rabbinowitsch, E. | Steffen, D. | Sanders, M. | Ma, J. | Kohara, Y. | Sharp, S. | Simmonds, M. | Spiegler, S. | Tivey, A. | Sugano, S. | White, B. | Walker, D. | Woodward, J. | Winckler, T. | Tanaka, Y. | Shaulsky, G. | Schleicher, M. | Weinstock, G. | Rosenthal, A. | Cox, E.C. | Chisholm, R. L. | Gibbs, R. | Loomis, W. F. | Platzer, M. | Kay, R. R. | Williams, J. | Dear, P. H. | Noegel, A. A. | Barrell, B. | Kuspa, A.
Nature  2005;435(7038):43-57.
The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes encode ~12,500 predicted proteins, a high proportion of which have long repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal rDNA element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal/fungal lineage after the plant/animal split, but Dictyostelium appears to have retained more of the diversity of the ancestral genome than either of these two groups.
PMCID: PMC1352341  PMID: 15875012
6.  Translation of NRF mRNA Is Mediated by Highly Efficient Internal Ribosome Entry 
Molecular and Cellular Biology  2000;20(8):2755-2759.
The ubiquitous transcription factor NRF (NF-κB repressing factor) is a constitutive transcriptional silencer of the multifunctional cytokine interferon-β. NRF mRNA contains a long 5′ untranslated region (5′UTR) predicted to fold into a strong secondary structure. The presence of stable hairpins is known to be incompatible with efficient translation by ribosomal scanning. Using dicistronic reporter gene constructs, we show that the NRF 5′UTR acts as an internal ribosome entry site (IRES) which directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES in various cell lines is at least 30-fold higher than that of picornaviral IRESs. The NRF 5′UTR also functions as a translational enhancer in the context of monocistronic mRNAs. Our results indicate that the NRF 5′UTR contains a highly potent IRES, which may allow for an alternate mode of translation under physiological conditions in which cap-dependent translation is inhibited.
PMCID: PMC85491  PMID: 10733578
7.  Preoperative diagnosis of a pulmonary artery sarcoma. 
Thorax  1995;50(9):1014-1017.
A pulmonary artery sarcoma was diagnosed preoperatively by magnetic resonance imaging enhanced with gadolinium and confirmed by percutaneous computed tomographic guided needle biopsy. Accurate preoperative diagnosis allowed planned curative surgery with removal of the right ventricular outflow tract and reconstructive surgery using a cryopreserved homograft.
PMCID: PMC1021323  PMID: 8539663
8.  On the use of double FLP recognition targets (FRTs) in the LTR of retroviruses for the construction of high producer cell lines. 
Nucleic Acids Research  1996;24(9):1616-1624.
A pilot experiment for the construction of a hamster derived, high producer cell line using site specific recombination is described. In the experiment chromosomal loci with intrinsic high expression characteristics were sought via infection with a retroviral construct, containing double FRT sites and subsequent screening for overproduction of an encoded markergene. These sites were then targeted with a second vector, that recombined via the FLP/FRT system from Saccharomyces cerevisiae yielding cells that had the second construct at exactly the same position as the first. By using retroviral vectors with double and single FRT sites, respectively, stable clones can be created that can no longer be excised with FLP.
PMCID: PMC145857  PMID: 8649977
9.  The sequences of and distance between two cis-acting signals determine the efficiency of ribosomal frameshifting in human immunodeficiency virus type 1 and human T-cell leukemia virus type II in vivo. 
Journal of Virology  1994;68(9):6087-6091.
We have analyzed in cell culture the sequence elements that control the level of ribosomal frameshifting in the human T-cell leukemia virus type II (HTLV-2) gag-pro junction. The slippery sequence of HTLV-2 is sufficient to dictate a basal level of frameshifting. This level is enhanced by its upstream sequence context and by the downstream stem-loop structure which is located at an optimal distance of 7 bases. Frameshifting in human immunodeficiency virus gag-pol is similar to that of HTLV-2 gag-pro. However, experiments using hybrid cassettes of HTLV-2 and human immunodeficiency virus type 1 frameshift elements show that while the slippery sequence of HTLV-2 is less efficient, the stem-loop structure is a more efficient enhancer.
PMCID: PMC237019  PMID: 8057488
10.  Screening retroviral packaging cells for highly efficient virus production by using a combined selection procedure. 
Journal of Virology  1994;68(1):566-569.
To facilitate the screening for clones of transfected packaging cells producing a high yield of recombinant retrovirus, we present a fast and simple method for the isolation of overexpressing cells. By this method the efficiency of virus production can generally be enhanced 10- to 100-fold by application of high selection pressure. Cell lines which exhibit titers of up to 10(8) CFU/ml were obtained.
PMCID: PMC236323  PMID: 8254773
11.  A heptanucleotide sequence mediates ribosomal frameshifting in mammalian cells. 
Journal of Virology  1993;67(9):5579-5584.
Ribosomal frameshifting is an essential requirement for replication of many viruses and retrovirus-like elements. It is regarded as a potential target for antiretroviral therapy. It has been shown that the frameshifting event takes place in the -1 direction within a sequence, the slippery sequence, which is usually followed by structured RNA. To distinguish between the basic sequence requirements and the modulating elements in intact cells, we have established a sensitive assay system for quantitative determination of ribosomal frameshifting in mammalian cell culture. In this assay system, the gag and pol genes of human immunodeficiency virus type 1 are replaced by the genes for the functional enzymes beta-galactosidase and luciferase, respectively. The sensitivity of the test system allows us to demonstrate for the first time that the slippery sequence, a heptanucleotide, is sufficient to mediate a basal level of ribosomal frameshifting independent of its position within a gene. The stem-loop sequence serves only as a positive modulator. These data indicate that frameshifting could also occur during translation of cellular genes in which a slippery sequence is present within the reading frame. The resulting putative transframe proteins might have a functional importance for cellular processes.
PMCID: PMC237961  PMID: 8350413
12.  DNA-mediated gene transfer in Friend leukemia cells by cotransfection of simian virus 40 DNA with herpes simplex virus thymidine kinase DNA. 
Journal of Virology  1983;45(1):375-382.
Thymidine kinase-negative Friend leukemia cells were cotransfected with simian virus 40 (SV40) DNA and thymidine kinase gene DNA of herpes simplex virus type 1. The transfected thymidine kinase-positive cells were selected in HAT medium, and SV40 T-antigen expression was observed over many months in cells cultured under selective conditions, and after adaptation to normal growth medium under nonselective conditions. It was shown by Southern blot hybridization that SV40 DNA was integrated in multiple copies in the chromosomal DNA of several clones. All SV40 DNA-containing Friend leukemia cell clones analyzed were able to undergo induced erythroid differentiation. Induced cultures still expressed SV40 T-antigen to the same extent that untreated control cultures did.
PMCID: PMC256419  PMID: 6296444
13.  Gene shuttling: moving of cloned DNA into and out of eukaryotic cells. 
Nucleic Acids Research  1982;10(4):1243-1256.
Successful shuttling of cloned DNA in eukaryotic cells should allow isolation of expressed genes. We tested the utility of cosmids for moving DNA into and out of eukaryotic cells. The unique cleavage of DNA at the cos site by the terminase function of lambda was exploited to maintain the linkage between the vector and inserted gene sequences, a prerequisite for successful rescue of the transforming DNA from high molecular weight DNA of the eukaryotic transformant. A cosmid recombinant containing the HSV thymidine kinase gene and a lambda recombinant containing the chicken thymidine kinase gene were used to test the feasability of this method. It was found that these recombinants can be rescued with high efficiency from DNA of HAT-resistant cells.
PMCID: PMC320522  PMID: 6280136
14.  Xeroradiographic techniques applied to assessment of Achilles tendon in inflammatory or metabolic diseases. 
Annals of the Rheumatic Diseases  1975;34(6):479-488.
Ten patients with inflammatory disease (rheumatoid arthritis, ankylosing spondylitis, Reiter's disease) or metabolic disease (gout, pseudogout, tendinous xanthomatosis) affecting the Achilles tendons are presented and discussed. Radiological lateral views of heel were obtained with xeroradiographic techniques, which permitted the recording on the same image of details of both bone and soft tissue and the evaluation and quantification of the changes in the Achilles tendons. Xeroradiography seems to be a very suitable radiological technique for routine use in the evaluation and follow up of rheumatic diseases of the foot.
PMCID: PMC1006469  PMID: 1221936
15.  5'-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency. 
Nucleic Acids Research  1981;9(10):2251-2266.
A method for cloning mRNAs has been used which results in a high yield of recombinants containing complete 5'-terminal mRNA sequences. It is not dependent on self-priming to generate double-stranded DNA and therefore the S1 nuclease digestion step is not required. Instead, the cDNA is dCMP-tailed at its 3'-end with terminal deoxynucleotidyl transferase (TdT). The synthesis of the second strand is primed by oligo(dG) hybridized to the 3'-tail. Double-stranded cDNA is subsequently tailed with dCTP and annealed to dGMP-tailed vector DNA. This approach overcomes the loss of the 5'-terminal mRNA sequences and the problem of artifacts which may be introduced into cloned cDNA sequences. Chicken lysozyme cDNA was cloned into pBR322 by this procedure with a transformation efficiency of 5 x 10(3) recombinant clones per ng of ds-cDNA. Sequence analysis revealed that at least nine out of nineteen randomly isolated plasmids contained the entire 5'-untranslated mRNA sequence. The data strongly support the conclusion that the 5'-untranslated region of the lysozyme mRNA is heterogeneous in length.
PMCID: PMC326843  PMID: 6166921
16.  Interferon regulatory factor 1 (IRF-1) mediates cell growth inhibition by transactivation of downstream target genes. 
Nucleic Acids Research  1993;21(12):2881-2889.
Interferon regulatory factor 1 (IRF-1) is a DNA-binding factor which recognizes regulatory elements in the promoters of interferon (IFN)-beta and some IFN-inducible genes. We observed that expression of transfected murine IRF-1 in different mammalian cell lines leads to down-regulation or stop of proliferation depending on the extent of expression. Expression of fusion proteins composed of IRF-1 and the hormone binding domain of the human estrogen receptor does not exhibit IRF-1 activity in the absence of estrogen. However, after estrogen treatment of the cells IFN-beta promoters are activated and the cells stop growing. As shown by expression of IRF-1 mutants both functions of the IRF-1-protein require DNA-binding and transcriptional activation. Since secreted factors including IFNs are not responsible for the anti-proliferative effect of IRF-1 we suggest that IRF-1 may be regarded as a negative regulator of cell growth which acts by activation of down-stream effector genes.
PMCID: PMC309674  PMID: 8332497
18.  The actin-related protein Act3p of Saccharomyces cerevisiae is located in the nucleus. 
Molecular Biology of the Cell  1995;6(10):1263-1270.
Actin-related proteins, a group of protein families that exhibit about 50% sequence identity among each other and to conventional actin, have been found in a variety of eukaryotic organisms. In the budding yeast Saccharomyces cerevisiae, genes for one conventional actin (ACT1) and for three actin-related proteins (ACT2, ACT3, and ACT5) are known. ACT3, which we recently discovered, is an essential gene coding for a polypeptide of 489 amino acids (Act3p), with a calculated molecular mass of 54.8 kDa. Besides its homology to conventional actin, Act3p possesses a domain exhibiting weak similarity to the chromosomal protein HMG-14 as well as a potential nuclear localization signal. An antiserum prepared against a specific segment of the ACT3 gene product recognizes a polypeptide band of approximately 55 kDa in yeast extract. Indirect immunofluorescence experiments with this antiserum revealed that Act3p is located in the nucleus. Nuclear staining was observed in all cells regardless of the stage of the cell cycle. Independently, immunoblotting experiments with subcellular fractions showed that Act3p is indeed highly enriched in the nuclear fraction. We suggest that Act3p is an essential constituent of yeast chromatin.
PMCID: PMC301286  PMID: 8573785

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