scholarly journals A transgenic pig model expressing a ZsGreen1 reporter across an extensive array of tissues

2018 ◽  
Author(s):  
Amy T. Desaulniers ◽  
Rebecca A. Cederberg ◽  
Elizabeth P. Carreiro ◽  
Channabasavaiah B. Gurumurthy ◽  
Brett R. White
Keyword(s):  
Transgene Expression ◽  
Specific Activity ◽  
Integration Site ◽  
Expression Patterns ◽  
Genetically Engineered ◽  
Cmv Promoter ◽  
Tissue Samples ◽  
Tissue Specific ◽  
Wide Range ◽  
Agricultural Industries

ABSTRACTBackgroundThe advent of genetically engineered pig production has revealed a wide array of opportunities to enhance both biomedical and agricultural industries. One powerful method to develop these models is transgenesis; however, selection of a suitable promoter to drive transgene expression is critical. The cytomegalovirus (CMV) promoter is the most commonly used viral promoter as it robustly drives transgene expression in a ubiquitous nature. However, recent reports suggest that the level of CMV promoter activity is tissue-dependent in the pig. Therefore, the objective of this study was to quantify the activity of the CMV promoter in a wide range of porcine tissues. Swine harboring a CMV-ZsGreen1 transgene with a single integration site were utilized for this study. Thirty five tissue samples were collected from neonatal hemizygous (n = 3) and homozygous (n = 3) transgenic piglets and analyzed for ZsGreen1 abundance via immunoblot.ResultsZsGreen1 was detected in all tissues examined; however, quantification revealed that ZsGreen1 protein levels were tissue-specific. Within organs of the digestive system, for example, ZsGreen1 was most abundant in the salivary gland, moderately produced in the esophagus and levels were lowest in the stomach. Interestingly, abundance of ZsGreen1 also differed within organ. For instance, levels were highest in the right ventricle compared with other chambers of the heart. There was no effect of transgene dose as ZsGreen1 expression patterns were similar between homozygous and hemizygous piglets.ConclusionsUltimately, these results elucidate the tissue-specific activity of the CMV promoter in the neonatal pig. Moreover, this model can serve as a useful tool for research applications requiring reporter gene activity in mammalian organs.

scholarly journals Evaluation of Plant-Derived Promoters for Constitutive and Tissue-Specific Gene Expression in Potato

Plants ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1520
Author(s):  
Dmitry Miroshnichenko ◽  
Aleksey Firsov ◽  
Vadim Timerbaev ◽  
Oleg Kozlov ◽  
Anna Klementyeva ◽  
...  
Keyword(s):  
Gene Expression ◽  
Specific Activity ◽  
Expression Patterns ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Promoter Strength ◽  
Tissue Specific ◽  
Camv 35S

Various plant-derived promoters can be used to regulate ectopic gene expression in potato. In the present study, four promoters derived from the potato genome have been characterized by the expression of identical cassettes carrying the fusion with the reporter β-glucuronidase (gusA) gene. The strengths of StUbi, StGBSS, StPat, and StLhca3 promoters were compared with the conventional constitutive CaMV 35S promoter in various organs (leaves, stems, roots, and tubers) of greenhouse-grown plants. The final amount of gene product was determined at the post-transcriptional level using histochemical analysis, fluorometric measurements, and Western blot analysis. The promoter strength comparison demonstrated that the StUbi promoter generally provided a higher level of constitutive β-glucuronidase accumulation than the viral CaMV 35S promoter. Although the StLhca3 promoter was predominantly expressed in a green tissue-specific manner (leaves and stems) while StGBSS and StPat mainly provided tuber-specific activity, a “promoter leakage” was also found. However, the degree of unspecific activity depended on the particular transgenic line and tissue. According to fluorometric data, the functional activity of promoters in leaves could be arranged as follows: StLhca3 > StUbi > CaMV 35S > StPat > StGBSS (from highest to lowest). In tubers, the higher expression was detected in transgenic plants expressing StPat-gusA fusion construct, and the strength order was as follows: StPat > StGBSS > StUbi > CaMV 35S > StLhca3. The observed differences between expression patterns are discussed considering the benefits and limitations for the usage of each promoter to regulate the expression of genes in a particular potato tissue.

scholarly journals Locus Control Region of the Human CD2Gene in a Lentivirus Vector Confers Position-Independent Transgene Expression

2001 ◽  
Vol 75 (10) ◽  
pp. 4641-4648 ◽  
Author(s):  
Claudia M. Kowolik ◽  
Jun Hu ◽  
Jiing-Kuan Yee
Keyword(s):  
T Cell ◽  
Copy Number ◽  
Transgene Expression ◽  
Integration Site ◽  
Leukemia Virus ◽  
Globin Gene ◽  
Clonal Variation ◽  
Control Vector ◽  
Fourfold Increase ◽  
Wide Range

ABSTRACT Vectors derived from murine leukemia virus (MLV) have been used in many human gene therapy clinical trials. However, insertion of the locus control regions (LCRs) derived from the β-globin gene locus or the CD2 gene into MLV vectors frequently led to vector rearrangement. Since the human immunodeficiency virus (HIV) sequence diverges significantly from the MLV sequence, we tested whether the LCR sequence is more stable in the context of an HIV vector. Clones derived from human fibrosarcoma line HT1080 cells transduced with an HIV vector containing the T-cell-specific CD2 LCR exhibit the same wide range of transgene expression as clones lacking the LCR. In contrast, Jurkat and primary T-cell clones derived from the transduction of the LCR-containing vector show, on average, a three- to fourfold increase in transgene expression relative to that of the control vector. This is consistent with previous observations that the CD2 LCR contains a T-cell-specific enhancer. In addition, the clones derived from the LCR-containing vector have a much lower clonal variation in transgene expression than those derived from the control vector. We also demonstrate that the level of transgene expression is proportional to the vector copy number. These results suggest that the human CD2 LCR sequence is compatible with HIV vector sequences and confers enhanced integration site-independent and copy number-dependent expression of the transgene. Thus, HIV vectors may represent the ideal vehicle to deliver genes controlled by various cis-acting elements such as LCRs.

scholarly journals Establishing a canine brain and tissue bank – molecular validation by RT-qPCR targeting three reference genes

2019 ◽  
Author(s):  
Sára Sándor ◽  
Kitti Tátrai ◽  
Kálmán Czeibert ◽  
Enikő Kubinyi
Keyword(s):  
Reference Genes ◽  
Expression Patterns ◽  
Tissue Bank ◽  
Molecular Techniques ◽  
Tissue Samples ◽  
Pet Dogs ◽  
Medical Background ◽  
Canine Brain ◽  
Age Related ◽  
Wide Range

Abstract Background: Dogs ( Canis familiaris ) are natural models of several human diseases, including age-related dementia. However, the molecular techniques, which are routinely applied in invertebrate and rodent models to study disease pathologies and mechanisms, has limited applicability in dogs, mainly because of ethical reasons. In the case of humans, the limited accessibility of tissue samples is at least partly solved by biobanks, which collect and store tissues and organs from voluntary donations. A similar approach with pet dogs could support both translational and veterinary research goals by providing access to good quality biological materials obtained from a wide range of dogs with known ancestry, life history and medical background. Therefore, we have established an initiative, the Canine Brain and Tissue Bank, to collect and store biological samples from pet dogs. Objectives: The molecular qualities of tissue samples collected and stored on a tissue bank are crucial for reliable downstream applications. We used quantitative Real-Time PCR methodology to assess the stability of mRNA content in our stored samples. Three previously validated reference genes, GAPDH , HMBS and HPRT1 were chosen as targets. Results: The tested reference genes showed expression patterns and stability values that were consistent with the literature. Conclusions: Based on the results, the molecular quality of tissues collected in the CBTB’s donations system can fit in the standards of dog gene expression analyses.

scholarly journals Atlas of tissue-specific and tissue-preferential gene expression in ecologically and economically significant conifer Pinus sylvestris

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11781
Author(s):  
Sandra Cervantes ◽  
Jaana Vuosku ◽  
Tanja Pyhäjärvi
Keyword(s):  
Gene Expression ◽  
Pinus Sylvestris ◽  
Tissue Specificity ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Biological Databases ◽  
Ploidy Levels ◽  
Tissue Specific ◽  
Genomic Resources ◽  
Wide Range

Despite their ecological and economical importance, conifers genomic resources are limited, mainly due to the large size and complexity of their genomes. Additionally, the available genomic resources lack complete structural and functional annotation. Transcriptomic resources have been commonly used to compensate for these deficiencies, though for most conifer species they are limited to a small number of tissues, or capture only a fraction of the genes present in the genome. Here we provide an atlas of gene expression patterns for conifer Pinus sylvestris across five tissues: embryo, megagametophyte, needle, phloem and vegetative bud. We used a wide range of tissues and focused our analyses on the expression profiles of genes at tissue level. We provide comprehensive information of the per-tissue normalized expression level, indication of tissue preferential upregulation and tissue-specificity of expression. We identified a total of 48,001 tissue preferentially upregulated and tissue specifically expressed genes, of which 28% have annotation in the Swiss-Prot database. Even though most of the putative genes identified do not have functional information in current biological databases, the tissue-specific patterns discovered provide valuable information about their potential functions for further studies, as for example in the areas of plant physiology, population genetics and genomics in general. As we provide information on tissue specificity at both diploid and haploid life stages, our data will also contribute to the understanding of evolutionary rates of different tissue types and ploidy levels.

scholarly journals Position-specific activity of the Hox1.1 promoter in transgenic mice

Development ◽  
1990 ◽  
Vol 108 (3) ◽  
pp. 435-442 ◽  
Author(s):  
A.W. Puschel ◽  
R. Balling ◽  
P. Gruss
Keyword(s):  
Transgenic Mice ◽  
Transgene Expression ◽  
Hox Genes ◽  
Specific Activity ◽  
Regulatory Element ◽  
Single Cells ◽  
Expression Patterns ◽  
Positional Information ◽  
Promoter Sequences ◽  
Second Period

During development, positional values have to be assigned to groups of cells. The murine Hox genes are a class of genes that are predicted to be involved at some stage in this process. During embryogenesis they are expressed in distinct overlapping region- and stage-specific patterns and therefore must be regulated in response to positional information. In this study, we have analysed the activity of Hox1.1 promoter sequences in transgenic mice. The use of lacZ as a marker allows a detailed analysis of expression at the single cell level during early embryonic development. We show that 3.6 kbp of promoter and 1.7 kbp of 3′ sequences provide sufficient regulatory information to express a transgene in a spatial and temporal manner indistinguishable from the endogenous Hox1.1 gene during the period of development when Hox1.1 expression is established. The activation occurs in a strict order in specific ectodermal and mesodermal domains. Within each of these domains the transgene is activated over a period of four hours apparently randomly in single cells. In a following second period, Hox1.1 and transgene expression patterns diverge. In this period, transgene expression persists in many mesodermally derived cells that do not express Hox1.1 indicating the absence of a negative regulatory element in the transgene. The anterior boundary of transgene expression is identical to that of Hox1.1. However, no posterior boundary of transgene expression is set, suggesting that a separate element absent from the transgene specifies this boundary.

scholarly journals Comparative Transcriptome Analysis of Alpinia oxyphylla reveals Tissue-specific Expression of Flavonoid Biosynthesis Genes

2020 ◽  
Author(s):  
Bingmiao Gao
Keyword(s):  
Transcriptome Analysis ◽  
Molecular Mechanisms ◽  
Expression Patterns ◽  
Genetically Engineered ◽  
Flavonoid Biosynthesis ◽  
Specific Gene ◽  
Specific Expression ◽  
Tissue Specific ◽  
Tissue Specific Expression ◽  
Alpinia Oxyphylla

Abstract Background: Alpinia oxyphylla is an important edible and medicinal herb, and its dried fruits are widely used in traditional herbal medicine. Flavonoids are one of the main chemical compounds in A. oxyphylla ; however, the genetic and molecular mechanisms of flavonoid biosynthesis are not well understood. Methods: We performed transcriptome analysis in the fruit, root, and leaf tissues of A. oxyphylla to delineate tissue-specific gene expression and metabolic pathways in this medicinal plant. Results: In all, 8.85, 10.10, 8.68, 6.89, and 8.51 Gb clean data were obtained for early-, middle-, and late-stage fruits, leaves, and roots, respectively. Furthermore, 50,401 unigenes were grouped into functional categories based on four databases, namely Nr (47,745 unigenes), Uniprot (49,685 unigenes), KOG (20,153 unigenes), and KEGG (27,285 unigenes). A total of 3,110 differentially expressed genes and five distinct clusters with similar expression patterns were obtained, in which 27 unigenes encoded 13 key enzymes (such as CHS, CHI, F3H, FLS, ANS ) associated with flavonoid biosynthesis. Conclusion: The tissue-specific expression of the genes corresponds to accumulation of flavonoids in these tissues.These results provide insights into the molecular mechanism of flavonoid biosynthesis in A. oxyphylla and application of genetically engineered varieties of A. oxyphylla .

scholarly journals Atlas of tissue-specific and tissue-preferential gene expression in ecologically and economically significant conifer Pinus sylvestris

2020 ◽  
Author(s):  
Sandra Cervantes ◽  
Jaana Vuosku ◽  
Dorota Paczesniak ◽  
Tanja Pyhäjärvi
Keyword(s):  
Gene Expression ◽  
Pinus Sylvestris ◽  
Tissue Specificity ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Biological Databases ◽  
Ploidy Levels ◽  
Tissue Specific ◽  
Genomic Resources ◽  
Wide Range

AbstractDespite their ecological and economical importance, conifers genomic resources are limited, mainly due to the large size and complexity of their genomes. Additionally, the available genomic resources lack complete structural and functional annotation. Transcriptomic resources have been commonly used to compensate for these deficiencies, though for most conifer species they are limited to a small number of tissues, or capture only a fraction of the genes present in the genome.Here we provide an atlas of gene expression patterns for conifer Pinus sylvestris across five tissues: embryo, megagametophyte, needle, phloem, and vegetative bud. We used a wide range of tissues and focused our analyses on the expression profiles of genes at tissue level. We provide comprehensive information of the per-tissue normalized expression level, indication of tissue preferential upregulation and tissue-specificity of expression. We identified a total of 48,001 tissue preferentially upregulated and tissue specifically expressed genes, of which 28% have annotation in the Swiss-Prot database. Even though most of the putative genes identified do not have functional information in current biological databases, the tissue-specific patterns discovered provide valuable information about their potential functions for further studies, as for example in the areas of plant physiology, population genetics, and genomics in general. As we provide information on tissue specificity at both diploid and haploid life stages, our data will also contribute to the understanding of evolutionary rates of different tissue types and ploidy levels.

scholarly journals Conservation of expression regulation throughout the animal kingdom

2014 ◽  
Author(s):  
Michael Kuhn ◽  
Andreas Beyer
Keyword(s):  
Gene Expression ◽  
Related Species ◽  
Expression Patterns ◽  
Gene Families ◽  
Tissue Expression ◽  
Rate Of Change ◽  
Gene Expression Patterns ◽  
Transfer Of Knowledge ◽  
Tissue Specific ◽  
Wide Range

Following the increase in available sequenced genomes, tissue-specific transcriptomes are being determined for a rapidly growing number of highly diverse species. Traditionally, only the transcriptomes of related species with equivalent tissues have been compared. Such an analysis is much more challenging over larger evolutionary distances when complementary tissues cannot readily be defined. Here, we present a method for the cross-species mapping of tissue-specific and developmental gene expression patterns across a wide range of animals, including many non-model species. Our approach maps gene expression patterns between species without requiring the definition of homologous tissues. With the help of this mapping, gene expression patterns can be compared even across distantly related species. In our survey of 36 datasets across 27 species, we detected conserved expression programs on all taxonomic levels, both within animals and between the animals and their closest unicellular relatives, the choanoflagellates. We found that the rate of change in tissue expression patterns is a property of gene families. Our findings open new avenues of study for the comparison and transfer of knowledge between different species.

Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity

2007 ◽  
Vol 292 (3) ◽  
pp. C1024-C1032 ◽  
Author(s):  
Ketrija Touw ◽  
April M. Hoggatt ◽  
Gina Simon ◽  
B. Paul Herring
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Transgene Expression ◽  
Promoter Activity ◽  
Specific Activity ◽  
Integration Site ◽  
Ectopic Expression ◽  
Muscle Cells ◽  
Insulator Elements

Mouse telokin and SM22α promoters have previously been shown to direct smooth muscle cell-specific expression of transgenes in vivo in adult mice. However, the activity of these promoters is highly dependent on the integration site of the transgene. In the current study, we found that the ectopic expression of telokin promoter transgenes could be abolished by flanking the transgene with insulator elements from the H19 gene. However, the insulator elements did not increase the proportion of mouse lines that exhibited consistent, detectable levels of transgene expression. In contrast, when transgenes were targeted to the hprt locus, both telokin and SM22α promoters resulted in reproducible patterns and levels of transgene expression in all lines of mice examined. Telokin promoter transgene expression was restricted to smooth muscle tissues in adult and embryonic mice. As reported previously, SM22α transgenes were expressed at high levels specifically in arterial smooth muscle cells; however, in contrast to randomly integrated transgenes, the hprt-targeted SM22α transgenes were also expressed at high levels in smooth muscle cells in veins, bladder, and gallbladder. Using hprt-targeted transgenes, we further analyzed elements within the telokin promoter required for tissue specific activity in vivo. Analysis of these transgenes revealed that the CArG element in the telokin promoter is required for promoter activity in all tissues and that the CArG element and adjacent AT-rich region are sufficient to drive transgene expression in bladder but not intestinal smooth muscle cells.

scholarly journals Comparative transcriptome analysis of Alpinia oxyphylla Miq. reveals tissue-specific expression of flavonoid biosynthesis genes

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Lin Yuan ◽  
Kun Pan ◽  
Yonghui Li ◽  
Bo Yi ◽  
Bingmiao Gao
Keyword(s):  
Transcriptome Analysis ◽  
Molecular Mechanisms ◽  
Expression Patterns ◽  
Genetically Engineered ◽  
Flavonoid Biosynthesis ◽  
Specific Gene ◽  
Specific Expression ◽  
Tissue Specific ◽  
Alpinia Oxyphylla ◽  
Leaf Tissues

Abstract Background Alpinia oxyphylla Miq. is an important edible and medicinal herb, and its dried fruits are widely used in traditional herbal medicine. Flavonoids are one of the main chemical compounds in A. oxyphylla; however, the genetic and molecular mechanisms of flavonoid biosynthesis are not well understood. We performed transcriptome analysis in the fruit, root, and leaf tissues of A. oxyphylla to delineate tissue-specific gene expression and metabolic pathways in this medicinal plant. Results In all, 8.85, 10.10, 8.68, 6.89, and 8.51 Gb clean data were obtained for early-, middle-, and late-stage fruits, leaves, and roots, respectively. Furthermore, 50,401 unigenes were grouped into functional categories based on four databases, namely Nr (47,745 unigenes), Uniprot (49,685 unigenes), KOG (20,153 unigenes), and KEGG (27,285 unigenes). A total of 3110 differentially expressed genes (DEGs) and five distinct clusters with similar expression patterns were obtained, in which 27 unigenes encoded 13 key enzymes associated with flavonoid biosynthesis. In particular, 9 DEGs were significantly up-regulated in fruits, whereas expression of 11 DEGs were highly up-regulated in roots, compared with those in leaves. Conclusion The DEGs and metabolic pathway related to flavonoids biosynthesis were identified in root, leaf, and different stages of fruits from A. oxyphylla. These results provide insights into the molecular mechanism of flavonoid biosynthesis in A. oxyphylla and application of genetically engineered varieties of A. oxyphylla.

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