Arne Lund Jørgensen

Pig transgenesis by Sleeping Beauty DNA transposition

Modelling of human disease in genetically engineered pigs provides unique possibilities in biomedical research and in studies of disease intervention. Establishment of methodologies that allow efficient gene insertion by non-viral gene carriers is an important step towards development of new disease models. In this report, we present transgenic pigs created by Sleeping Beauty DNA transposition in primary porcine fibroblasts in combination with somatic cell nuclear transfer by handmade cloning. Göttingen minipigs expressing green fluorescent protein are produced by transgenesis with DNA transposon vectors carrying the transgene driven by the human ubiquitin C promoter. These animals carry multiple copies (from 8 to 13) of the transgene and show systemic transgene expression. Transgene-expressing pigs carry both transposase-catalyzed insertions and at least one copy of randomly inserted plasmid DNA. Our findings illustrate critical issues related to DNA transposon-directed transgenesis, including coincidental plasmid insertion and relatively low Sleeping Beauty transposition activity in porcine fibroblasts, but also provide a platform for future development of porcine disease models using the Sleeping Beauty gene insertion technology.

Identification and characterization of GFAPκ, a novel glial fibrillary acidic protein isoform

Glial fibrillary acidic protein (GFAP) is the principal component of the intermediary filaments in mature astrocytes of the central nervous system (CNS). The protein consists of three domains: the head, the coiled‐coil, and the tail. Here, we describe the isolation of an evolutionary conserved novel GFAP isoform, GFAPκ, produced by alternative splicing and polyadenylation of the 3′‐region of the human GFAP pre‐mRNA. As a consequence, the resulting human GFAPκ protein harbors a nonconserved C‐terminal tail sequence distinct from the tails of GFAPα, the predominant GFAP isoform, and GFAPε, an isoform which also results from alternative splicing. The head and coiled‐coil rod domains are identical between the three GFAP isoforms. Interestingly, GFAPκ is incapable of forming homomeric filaments, and increasing GFAPκ expression levels causes a collapse of intermediate filaments formed by GFAPα. In searching for a biological relevance of GFAPκ, we noticed that mRNA expression levels of GFAPα, GFAPε, and GFAPκ are gradually increased during development of the embryonic pig brain. However, whereas the GFAPα/GFAPε ratio is constant, the GFAPκ/GFAPε ratio decreases during brain development. Furthermore, in glioblastoma tumors, an increased GFAPκ/GFAPε ratio is detected. Our results suggest that the relative expression level of the GFAPκ isoform could modulate the properties of GFAP intermediate filaments and perhaps thereby influencing the motility of GFAP positive astrocytes and progenitor cells within the CNS.

Equal proportion of adult male and female homozygous for the 677C T mutation in the methylenetetrahydrofolate reductase polymorphism

Evidence suggests that a polymorphism of the autosomal gene encoding for 5,10 methylenetetrahydrofolate reductase (MTHFR) [Frosst et al., 1995] gives rise to a thermolabile form [Kang et al., 1988] of the enzyme that in the homozygous state is associated with increased levels of homocysteine under conditions of less than optimal folate nutrition [Jacques et al., 1996]. This polymorphism is due to a C!T substitution at nucleotide 677 which converts an alanine to valine in a conserved portion of themolecule [Frosst et al., 1995] and appears to be a risk factor for increased plasma levels of homocysteine and vascular diseases [Boushey et al., 1995; Jacques et al., 1996; Kluijtmans et al., 1997]. In a 1999 letter to the Editor of the American Journal of Medical Genetics, RimaRozen et al. presented data examining the frequencies of the three methylenetetrahydrofolate reductase (MTHFR) genotypes in newborn healthy infants ascertained by the California Birth Defects Monitoring Program [Rozen and Shaw, 1999]. It was suggested that homozygosity for the common 677 C!Tmutation inMTHFR genewasmore frequent among newbornmales than among newborn females. The apparent gender difference was observed among 915 newborn infants used as control populations in studies of an association between the MTHFR polymorphism and neural tubedefects.RozenandShaw [1999] stratified this groupof 915 newborn control healthy infants by MTHFR genotypes and found that among 150 infants who had the TT variant for MTHFR only 50 (33%) were female. This was significantly less (P< 0.01) than the expected 50% of female infants. Weexamined the frequencies of the threeMTHFRgenotypes in 559 healthy, Caucasian, male and female adult volunteers from Seattle, WA for a controlled intervention study of homocysteine response to folic acid supplementation with variable dosage. As shown in Table I, and similar to the findings of Rozen and Shaw [1999], we detected a significantly lower proportion of females than males to be homozygous for the 677C!Tmutation inMTHFR. Among the 173 males and 386 females analyzed, we found 13.9% of male volunteers were TT homozygotes in comparison to 7.8% of female volunteers (Chi square: P< 0.05). We also found that the T allele relative frequency is significantly different betweenmales and females (P1⁄4 0.02). These results suggest some variation in our data in the MTHFR genotypes due to gender. We hypothesized that theremay be two possible reasons for the observed reduction in frequency ofMTHFR TT homozygotes in females. The first is a possible prenatal selection against TT female homozygotes, and the second is a possible higher T allele frequency in males than females. To explore the observed reduction in frequency of MTHFR TT homozygotes in females, we conducted a literature search for studies specifying MTHFR genotypes and gender for healthy adult individuals, typically from population and case-control studies.Weassumed that a sex difference observed at birth would survive into adulthood since we know of no postnatal condition associated with the MTHFR polymorphism that would eliminate such differences during childhood. The keywords used to produce a primary list in the PubMed literature search were MTHFR, gender, and sex. Two articles were carefully reviewed for related articles [Slattery et al., 1999;Ulrich et al., 2000]. Fromthis search, the list of articles in Table II was generated and was considered representative of the literature on the subject [Adams et al., 1996; Brugada and Marian, 1997; Christensen et al., 1997; Galinsky et al., 1997; Heijmans et al., 1999; Slattery et al., 1999; Stegmann et al., 1999; Chango et al., 2000; Delvin et al., 2000; D’Angelo et al., 2000; Ho, 2000; Hustad et al., 2000; Ulrich et al., 2000; Dekou et al., 2001; Dierkes et al., 2001; Guillen et al., 2001; Kimura et al., 2001; Saw et al., 2001; Wu et al., 2001; Beresford, 2004 unpublished data]. Each article was examined for information onMTHFR genotypes and gender. Table II shows the numbers and percentages of males and females with TT homozygosity for the MTHFR 677 C!T polymorphism, the total number of males and females, age group, and geographic and ethnic origin for each study. We show race/ethnicity data because the incidence of this genetic polymorphism varies among populations.Weshowgeographic originbecause theallele frequencies of theMTHFR 677C!Tpolymorphism also vary significantly between populations from different geographic areas. For example, the T allele frequency varies from 22 to 45% between populations from northern and southern European countries, respectively [Andreassi et al., 2003]. Based on data from these studies, we show no consistent gender difference in homozygosity for the common 677 C!T mutation in MTHFR gene. If the studies are combined, as indicated at the bottom of Table II, the TT genotype frequency is almost the same in males and females (11.6 and 11.5%, respectively). Assessing only studies with a total of over 100 males and 100 females from Table II, similar results were obtained (data not shown). We also calculated the predicted distributions of the genotypes for each gender using the relative frequencies of the C and T allele and the Hardy– Weinberg equation. We found that the predicted distributions are in complete accord with the observed distributions shown in Table I. The principles of the Hardy–Weinberg equation apply in these data suggesting there is no prenatal selection Cheryl A.M. Anderson and Arne Lund Jorgensen completed the work while at the University of Washington, School of Public Health and Community Medicine (CA) and Division of Medical Genetics (ALJ).

Breakpoints in Robertsonian translocations are localized to satellite III DNA by fluorescence in situ hybridization.

We characterized 21 t(13;14) and 3 t(14;21) Robertsonian translocations for the presence of DNA derived from the short arms of the translocated acrocentric chromosomes and identified their centromeres. Nineteen of these 24 translocation carriers were unrelated. Using centromeric alpha-repeat DNA as chromosome-specific probe, we found by in situ hybridization that all 24 translocation chromosomes were dicentric. The chromatin between the two centomeres did not stain with silver, and no hybridization signal was detected with probes for rDNA or beta-satellite DNA that flank the distal and proximal ends of the rDNA region on the short arm of the acrocentrics. By contrast, all 24 translocation chromosomes gave a distinct hybridization signal when satellite III DNA was used as probe. This result strongly suggests that the chromosomal rearrangements leading to Robertsonian translocations occur preferentially in satellite III DNA. We hypothesize that guanine-rich satellite III repeats may promote chromosomal recombination by formation of tetraplex structures. The findings localize satellite III DNA to the short arm of the acrocentric chromosomes distal to centromeric alpha-repeat DNA and proximal to beta-satellite DNA.

Structural and functional characterization of the zebrafish gene for glial fibrillary acidic protein, GFAP

Glial fibrillary acidic protein, GFAP, is an astrocyte-specific member of the family of intermediate filament proteins which are involved in formation of the cytoskeletal structure. We here present a characterization of the zebrafish GFAP gene and corresponding protein. The zebrafish GFAP gene have the same exon-intron organization as the mammalian orthologoue genes. Comparison of the protein with mammalian GFAP shows that the amino acid sequence is highly conserved in the rod and tail domains whereas the head domain has diverged. Zebrafish GFAP exhibits functional characteristics of an intermediate filament protein such as dimerization potential, capacity to assembly into filaments, and cytoskeletal localization. Mutations in human GFAP have been associated with a severe childhood brain disorder called Alexander disease. Interestingly, the mutations affect preferentially amino acid residues of GFAP that are evolutionarily conserved. This indicates that a change of functionally core residues in GFAP is a prerequisite for the disease phenotype to develop and the initial steps in the pathogenesis may thus be modeled in zebrafish.

Regulatory mechanisms for 3′-end alternative splicing and polyadenylation of the Glial Fibrillary Acidic Protein, GFAP, transcript

The glial fibrillary acidic protein, GFAP, forms the intermediate cytoskeleton in cells of the glial lineage. Besides the common GFAPα transcript, the GFAPε and GFAPκ transcripts are generated by alternative mRNA 3′-end processing. Here we use a GFAP minigene to characterize molecular mechanisms participating in alternative GFAP expression. Usage of a polyadenylation signal within the alternatively spliced exon 7a is essential to generate the GFAPκ and GFAPκ transcripts. The GFAPκ mRNA is distinct from GFAPε mRNA given that it also includes intron 7a. Polyadenylation at the exon 7a site is stimulated by the upstream splice site. Moreover, exon 7a splice enhancer motifs supported both exon 7a splicing and polyadenylation. SR proteins increased the usage of the exon 7a polyadenylation signal but not the exon 7a splicing, whereas the polypyrimidine tract binding (PTB) protein enhanced both exon 7a polyadenylation and exon 7a splicing. Finally, increasing transcription by the VP16 trans-activator did not affect the frequency of use of the exon 7a polyadenylation signal whereas the exon 7a splicing frequency was decreased. Our data suggest a model with the selection of the exon 7a polyadenylation site being the essential and primary event for regulating GFAP alternative processing.

A new mechanism in blue cone monochromatism.

Abstract Blue cone monochromatism (BCM) is a rare X-linked colour vision disorder characterized by the absence of both red and green cone sensitivity. Most mutations leading to BCM fall into two classes of alterations in the red and green pigment gene array at Xq28. In one class the red and green pigment genes are inactivated by deletion in the locus control region. In the second class genetic rearrangements have created an isolated pigment gene that carries an inactivating point mutation. Here we describe a clinical case of BCM caused by a new mutation where exon 4 of an isolated red pigment gene has been deleted. The finding represents the first intragenic deletion yet described among red and green pigment genes.

Generation of minipigs with targeted transgene insertion by recombinase-mediated cassette exchange (RMCE) and somatic cell nuclear transfer (SCNT)

Targeted transgenesis using site-specific recombinases is an attractive method to create genetically modified animals as it allows for integration of the transgene in a pre-selected transcriptionally active genomic site. Here we describe the application of recombinase-mediated cassette exchange (RMCE) in cells from a Göttingen minipig with four RMCE acceptor loci, each containing a green fluorescence protein (GFP) marker gene driven by a human UbiC promoter. The four RMCE acceptor loci segregated independent of each other, and expression profiles could be determined in various tissues. Using minicircles in RMCE in fibroblasts with all four acceptor loci and followed by SCNT, we produced piglets with a single copy of a transgene incorporated into one of the transcriptionally active acceptor loci. The transgene, consisting of a cDNA of the Alzheimer’s disease-causing gene PSEN1M146I driven by an enhanced human UbiC promoter, had an expression profile in various tissues similar to that of the GFP marker gene. The results show that RMCE can be done in a pre-selected transcriptionally active acceptor locus for targeted transgenesis in pigs.

Efficiency of two enucleation methods connected to handmade cloning to produce transgenic porcine embryos.

The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41-42 h oocytes maturation, the oocytes were further cultured with or without 0.4 microg/ml demecolcine for 45 min [chemically assisted handmade enucleation (CAHE) group vs polar body (PB) oriented handmade enucleation (OHE) group respectively]. After removal of the cumulus cells and partial digestion of the zona pellucida, oocytes with visible extrusion cones and/or polar bodies attached to the surface were subjected to oriented bisection. Putative cytoplasts without extrusion cones or PB were selected as recipients. Two cytoplasts were electrofused with one transgenic fibroblasts expressing green fluorescent protein (GFP), while non-transgenic fibroblasts were used as controls. Reconstructed embryos were cultured in Well of Wells (WOWs) with porcine zygote medium 3 (PZM-3) after activation. Cleavage and blastocyst rates were registered on day 2 and day 7 of in vitro culture respectively. Meanwhile, the total blastocyst cell number was counted on day 7. We found that the difference was only observed between blastocyst rates (38.6 +/- 2% vs 48.1 +/- 3%) of cloned embryos with GFP transgenic fibroblast cells after CAHE vs OHE. With adjusted time-lapse for zonae-free cloned embryos cultured in WOWs with PZM-3, it was obvious that in vitro developmental competence after CAHE was compromised when compared with the OHE method. OHE enucleation method seems to be a potential superior alternative method used for somatic cell nuclear transfer (SCNT) with transgenic fibroblast cells.

Molecular characterization and temporal expression profiling of presenilins in the developing porcine brain

BackgroundThe transmembrane presenilin (PSEN) proteins, PSEN1 and PSEN2, have been proposed to be the catalytic components of the γ-secretase protein complex, which is an intramembranous multimeric protease involved in development, cell regulatory processes, and neurodegeneration in Alzheimers disease. Here we describe the sequencing, chromosomal mapping, and polymorphism analysis of PSEN1 and PSEN2 in the domestic pig (Sus scrofa domesticus).ResultsThe porcine presenilin proteins showed a high degree of homology over their entire sequences to the PSENs from mouse, bovine, and human. PSEN1 and PSEN2 transcription was examined during prenatal development of the brain stem, hippocampus, cortex, basal ganglia, and cerebellum at embryonic days 60, 80, 100, and 114, which revealed distinct temporal- and tissue-specific expression profiles. Furthermore, immunohistochemical analysis of PSEN1 and PSEN2 showed similar localization of the proteins predominantly in neuronal cells in all examined brain areas.ConclusionThe data provide evidence for structural and functional conservation of PSENs in mammalian lineages, and may suggest that the high sequence similarity and colocalization of PSEN1 and PSEN2 in brain tissue reflect a certain degree of functional redundancy. The data show that pigs may provide a new animal model for detailed analysis of the developmental functions of the PSENs.