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The expression of transgene loci in mammals often occurs in a heterocellular fashion resulting in variegated patterns of expression. We have examined the effect of chromosomal integration site, copy number, and transcriptionally activating sequences on the variegation of a keratin 5-lacZ (K5Z) construct in the stratified epithelia of transgenic mic...
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... It is possible, however, that these mechanisms may have played some role in determining the pattern of NEP expression in our DTs. Some of these mechanisms include mosaicism (Koetsier et al. 1996;Schonig et al. 2010), mutagenesis of the transgene or surrounding genes (Walters et al. 2009), epigenetic effects (Koetsier et al. 1996;Ramirez et al. 2001;Wang et al. 2009;Lorthongpanich et al. 2013), or species-specific codon bias (Plotkin and Kudla 2011). Also, the Rosa26 promoter itself, although originally characterized to drive high levels of universal expression (Kisseberth et al. 1999), may have been an inadvertent source of variability, or even tissue specificity (Wortge et al. 2010;Walters et al. 2009). ...
Neprilysin (NEP) is a cell surface metallopeptidase found in many tissues. Based mostly on pharmacological manipulations, NEP has been thought to protect blood vessels from plasma extravasation. We have suggested that NEP may protect against pulmonary vascular injury. However, these prior studies did not utilize mice which overexpress NEP. The aims of the present investigation were to develop and characterize doubly transgenic (DT) mice that overexpress NEP universally and conditionally, and to investigate the protective effect that overexpressed NEP may have against plasma extravasation in the vasculature. The duodenum, which is often used to assess vascular permeability, and in which the NEP protein was overexpressed in our DT mice two-fold, was selected as our experimental preparation. We found that substance P-induced plasma extravasation was decreased substantially (3.5-fold) in the duodenums of our doxycycline-treated DT mice, giving independent evidence of NEP’s protective effects against plasma extravasation. Transgenic lung NEP protein was not stably expressed in the DT mice, so we were not able to test the effect of NEP overexpression in the lung. Although initially overexpressed nearly nine-fold at that site, pulmonary NEP protein overexpression eventually dissipated. Surprisingly, at a time when there was no lung transgenic NEP protein overexpression, lung NEP mRNA expression was still increased 23-fold, indicating that the expression defect probably is not transcriptional. These studies help to characterize our complex transgenic model of NEP overexpression and further demonstrate NEP’s protective effects against plasma extravasation.
... In the past decades, the GE livestock have been commonly generated by either microinjection or transfection of expression cassettes into the animal genome, in which the transgenes insertion were essentially random (Laible et al. 2015a). The random integration possibly results in unpredictable expression of transgenes, insertional mutagenesis and unwanted pathological side effects (Clark et al. 1997;Dobie et al. 1996;Fattori et al. 1994;Gao et al. 2007; Lewis et al. 1993;Milot et al. 1996;Pedram et al. 2006;Ramirez et al. 2001). Although gene targeting by homologous recombination (HR) could overcome these shortcomings, the lower frequency of HR in somatic cells hampers its application in GE livestock. ...
With the technological development of several engineered endonucleases (EENs), such as zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9, gene targeting by homologous recombination has been efficiently improved to generate site-specifically genetically modified livestock. However, few studies have been done to investigate the health and fertility of these animals. The purpose of the present study is to investigate if gene targeting events and a recloning procedure would affect the production traits of EEN-mediated gene targeted bucks. TALEN-mediated β-lactoglobulin (BLG) gene mono-allelic knockout (BLG
+/−) goats and bi-allelic knockout (BLG
−/−) buck produced by using sequential gene targeting combined with recloning in fibroblasts from BLG
+/− buck were used to evaluate their health and fertility. Birth weight and postnatal growth of BLG
+/− bucks were similar to the wild-type goats. None of the parameters for both fresh and frozen-thawed semen quality were significantly different in BLG
+/− or BLG
−/− bucks compared to their corresponding comparators. In vitro fertilization (IVF) test revealed that the proportion of IVF oocytes developing to the blastocyst stage was identical among BLG
+/−, BLG
−/− and wild-type bucks. Conception rates of artificial insemination were respectively 42.3, 38.0 and 42.6 % for frozen-thawed semen from the BLG
+/−, BLG
−/− and wild-type bucks. In addition, germline transmission of the targeted BLG modification was in accordance with Mendelian rules. These results demonstrated that the analyzed growth and reproductive traits were not impacted by targeting BLG gene and recloning, implicating the potential for dairy goat breeding of BLG
+/− and BLG
−/− bucks.
... Chromosomal context is an important factor to consider when expressing heterologous genes, because promoter interference, chromatin structure or other epigenetic modifications can have a negative impact on gene activation 28,29 . Thus, we tested GFP expression from cassettes integrated at nine different chromosomal loci. ...
Current biopharmaceutical manufacturing systems are not compatible with portable or distributed production of biologics, as they typically require the development of single biologic-producing cell lines followed by their cultivation at very large scales. Therefore, it remains challenging to treat patients in short time frames, especially in remote locations with limited infrastructure. To overcome these barriers, we developed a platform using genetically engineered Pichia pastoris strains designed to secrete multiple proteins on programmable cues in an integrated, benchtop, millilitre-scale microfluidic device. We use this platform for rapid and switchable production of two biologics from a single yeast strain as specified by the operator. Our results demonstrate selectable and near-single-dose production of these biologics in <24 h with limited infrastructure requirements. We envision that combining this system with analytical, purification and polishing technologies could lead to a small-scale, portable and fully integrated personal biomanufacturing platform that could advance disease treatment at point-of-care.
... Therefore, the fidelity of reporter expression is limited only by the fidelity of Cre expression. This is in contrast to some transgenes and reporters (Ramírez et al., 2001). ...
Female eutherian mammals use X chromosome inactivation (XCI) to epigenetically regulate gene expression from ∼4% of the genome. To quantitatively map the topography of XCI for defined cell types at single cell resolution, we have generated female mice that carry X-linked, Cre-activated, and nuclear-localized fluorescent reporters-GFP on one X chromosome and tdTomato on the other. Using these reporters in combination with different Cre drivers, we have defined the topographies of XCI mosaicism for multiple CNS cell types and of retinal vascular dysfunction in a model of Norrie disease. Depending on cell type, fluctuations in the XCI mosaic are observed over a wide range of spatial scales, from neighboring cells to left versus right sides of the body. These data imply a major role for XCI in generating female-specific, genetically directed, stochastic diversity in eutherian mammals on spatial scales that would be predicted to affect CNS function within and between individuals.
... Many methods are available to produce transgenic donor cells, and the traditional method relies on random integration of the transgene of interest. However, random integration into chromosomes suffers from low stable integration [7], and variable expression levels of the genes due to positional effects and the number of inserted copies891011. Homologous recombination provides site specificity, but at a very low efficiency [12]. Furthermore, many virus-based gene transfer approaches are limited by their preference for integration into the gene-coding region [13,14], which is a safety risk of transgenic animal production. ...
Antibiotic selectable marker genes have been widely used to generate transgenic animals. Once transgenic animals have been obtained, the selectable marker is no longer necessary but raises public concerns regarding biological safety. The aim of this study was to prepare competent antibiotic selectable marker free transgenic cells for somatic cell nuclear transfer (SCNT). PhiC31 intergrase was used to insert a transgene cassette into a "safe harbor" in the bovine genome. Then, Cre recombinase was employed to excise the selectable marker under the monitoring of a fluorescent double reporter. By visually tracking the phenotypic switch from red to green fluorescence, antibiotic selectable marker free cells were easily detected and sorted by fluorescence-activated cell sorting. For safety, we used phiC31 mRNA and cell-permeant Cre protein in this study. When used as donor nuclei for SCNT, these safe harbor integrated marker-free transgenic cells supported a similar developmental competence of SCNT embryos compared with that of non-transgenic cells. After embryo transfer, antibiotic selectable marker free transgenic cattle were generated and anti-bacterial recombinant human β-defensin-3 in milk was detected during their lactation period. Thus, this approach offers a rapid and safe alternative to produce antibiotic selectable marker free transgenic farm animals, thereby making it a valuable tool to promote the healthy development and welfare of transgenic farm animals.
... Cell mosaicism, manifesting as expression of the transgene in some but not all cells, is a common phenomenon especially for transgenes directed by tissue-specific genes (see Discussion Serova et al. 2012). Some researchers have suggested that pericentromeric localization of transgenes prompts variegated expression (Dobie et al. 1996;Festenstein et al. 1996;Milot et al. 1996), although other studies do not support this notion (Ramirez et al. 2001). Absence of cell mosaicism in the mammary gland with pGoatcasGMCSF transgene expression is in accordance with an earlier study of transgenic mice carrying the human G-CSF gene under the control of the same promoter (Serova et al. 2012). ...
... In some cases, integration of the transgene near heterochromatin is accompanied by silencing due to a significant decrease in the proportion of cells that express the transgene (Dobie et al. 1996;Festenstein et al. 1996). However, cell mosaicism was observed in transgenic mice with transgene localization at pericentromeric and outside regions (Ramirez et al. 2001). For instance, the chicken lysozyme 5 0 MAR element fused with the keratin 5 promoter did not prevent cell mosaicism in stratified epithelia of mice with transgene localizations at pericentromeric and outside regions (Ramirez et al. 2001). ...
... However, cell mosaicism was observed in transgenic mice with transgene localization at pericentromeric and outside regions (Ramirez et al. 2001). For instance, the chicken lysozyme 5 0 MAR element fused with the keratin 5 promoter did not prevent cell mosaicism in stratified epithelia of mice with transgene localizations at pericentromeric and outside regions (Ramirez et al. 2001). ...
Expression of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene under the control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a matrix attachment region (MAR) element from the Drosophila histone 1 gene was studied in four and eight transgenic mouse lines, respectively. Of the four transgenic lines carrying the transgene without MAR, three had correct tissues-specific expression of the hGM-CSF gene in the mammary gland only and no signs of cell mosaicism. The concentration of hGM-CSF in the milk of transgenic females varied from 1.9 to 14 μg/ml. One line presented hGM-CSF in the blood serum, indicating ectopic expression. The values of secretion of hGM-CSF in milk of 6 transgenic lines carrying the transgene with MAR varied from 0.05 to 0.7 μg/ml, and two of these did not express hGM-CSF. Three of the four examined animals from lines of this group showed ectopic expression of the hGM-CSF gene, as determined by RT-PCR and immunofluorescence analyses, as well as the presence of hGM-CSF in the blood serum. Mosaic expression of the hGM-CSF gene in mammary epithelial cells was specific to all examined transgenic mice carrying the transgene with MAR but was never observed in the transgenic mice without MAR. The mosaic expression was not dependent on transgene copy number. Thus, the expected "protective or enhancer effect" from the MAR element on the hGM-CSF gene expression was not observed.
... These methods are followed by antibiotic selection of a stable pool of cells and functional screening to identify individual clones that have the correct function(s). However, random integration into chromosomes is inefficient [1], and the expression levels of genes vary greatly due to positional effects and the number of copies inserted [2,3,4,5]. As a result, the process of generating and selecting gene expression cells can be labor intensive and extremely time consuming. ...
The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.
... As well, the lack of correlation between pre-Cre excision-based expression of β-galactosidase and post-Cre-based expression of reporter constructs (18,39) is similar to our observed results where β-galactosidase expression showed more limited and mosaic expression compared with Cre-mediated expression of myc-Mdm4 protein and EGFP. These results may be attributable to elements within the β-geo cassette interfering with pCAGGs-based expression prior to Cre-excision and/or the fact that the bacterial β-galactosidase gene has previously been demonstrated to show mosaic expression particularly in adult tissues (41,42) that may be related to sub-optimal codon usage of the prokaryote sequences (18) or transgene silencing (41,42). ...
... As well, the lack of correlation between pre-Cre excision-based expression of β-galactosidase and post-Cre-based expression of reporter constructs (18,39) is similar to our observed results where β-galactosidase expression showed more limited and mosaic expression compared with Cre-mediated expression of myc-Mdm4 protein and EGFP. These results may be attributable to elements within the β-geo cassette interfering with pCAGGs-based expression prior to Cre-excision and/or the fact that the bacterial β-galactosidase gene has previously been demonstrated to show mosaic expression particularly in adult tissues (41,42) that may be related to sub-optimal codon usage of the prokaryote sequences (18) or transgene silencing (41,42). ...
The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3-4 weeks by using Gateway cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner.
... Thus, in our mice, the responder sequences might favour the attraction of repressing states in concomitance with an increase in copy numbers, leading to a minor number of expressing ORNs. The responder transgene sequences as the cause of the unexpected expression pattern: There is documented evidence that random integration of transgenes can fail to drive consistent exogenous expression due to several factors, such as genome position influences (Sabl and Henikoff 1996), mouse genetic background (Robertson et al. 2002; Opsahl et al. 2002; Padjen et al. 2005), number (Saveliev et al. 2003) and orientation (Stam et al. 1998), or sequence composition (Ramírez et al. 2001; Lotti et al. 2002) of transgene-item repeats, especially if viral sequences are included (Schumacher et al. 2000). Failure of tet-based strategies in vivo has also been reported. ...
In generating a conditional transgenic murine model based on a tetracycline-regulated system, we obtained unexpected patterns of expression due to the transcriptional inactivity of the tet-responder promoter. Here we show strong cell-type-restricted expression that was variegated to an extent determined by the number of responder transgene copies integrated into the host genome.
... It rather suggests a variegated expression of the TgTRSID transgene in the TgTRSID.41 line [27][28][29]. Overall, these data demonstrate that low levels of trans-repressor may be enough to ensure a knockout-like phenotype. ...
Spatial and temporal control of ovine prion protein (Prnp) gene expression was achieved in mice using two transgenes: a Prnp minigene with tet-operator sequences inserted 5' to exon 1 and a mouse neurofilament genomic clone carrying the chimeric-repressor TRSID cDNA. In bi-transgenic mice, ovine PrP(C) expression could be reversibly controlled in neuronal cells by doxycycline treatment whereas it remains constant in other cell types. Overall, this model opens opportunities to assess the involvement of cell types in prion diseases and PrP physiological function. It demonstrates the potentiality of the TRSID-silencer to precisely control temporal and spatial gene expression in vivo.
