scholarly journals Tamoxifen-inducible cardiac-specific Cre transgenic mouse using VIPR2 intron

2020 ◽  
Vol 36 (1) ◽  
Author(s):  
Hyun Jung Chin ◽  
So-young Lee ◽  
Daekee Lee
Keyword(s):  
Transgenic Mice ◽  
Transgenic Mouse ◽  
Gene Function ◽  
Regulatory Region ◽  
Genetically Engineered ◽  
Specific Gene ◽  
Specific Expression ◽  
Genetically Engineered Mouse Models ◽  
Reporter Mice ◽  
Vasoactive Intestinal Peptide Receptor

Abstract Genetically engineered mouse models through gene deletion are useful tools for analyzing gene function. To delete a gene in a certain tissue temporally, tissue-specific and tamoxifen-inducible Cre transgenic mice are generally used. Here, we generated transgenic mouse with cardiac-specific expression of Cre recombinase fused to a mutant estrogen ligand-binding domain (ERT2) on both N-terminal and C-terminal under the regulatory region of human vasoactive intestinal peptide receptor 2 (VIPR2) intron and Hsp68 promoter (VIPR2-ERT2CreERT2). In VIPR2-ERT2CreERT2 transgenic mice, mRNA for Cre gene was highly expressed in the heart. To further reveal heart-specific Cre expression, VIPR2-ERT2CreERT2 mice mated with ROSA26-lacZ reporter mice were examined by X-gal staining. Results of X-gal staining revealed that Cre-dependent recombination occurred only in the heart after treatment with tamoxifen. Taken together, these results demonstrate that VIPR2-ERT2CreERT2 transgenic mouse is a useful model to unveil a specific gene function in the heart.

Megakaryocyte gene targeting mediated by restricted expression of recombinase Cre

2011 ◽  
Vol 105 (01) ◽  
pp. 138-144 ◽  
Author(s):  
Adam Nowakowski ◽  
Sonia Alonso-Martín ◽  
Elena Arias-Salgado ◽  
Darío Fernández ◽  
MariPaz Vilar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Gene Targeting ◽  
Specific Gene ◽  
Specific Expression ◽  
Intense Staining ◽  
Hematopoietic Stem ◽  
Adult Mice ◽  
Reporter Mice

SummaryThe availability of mice with tissue-specific expression of recombinase Cre is the limiting step for a successful gene targeting by the Cre-LoxP methodology. This work aimed at generating transgenic mice with restricted expression of recombinase Cre in megakaryocytes and platelets, driven by the promoter of the αIIb gene (mαIIb-cre). Mice oocytes were microinjected with a 4.1 Kb construct comprising a 2.7 Kb promoter fragment of the glycoprotein αIIb gene, linked to the CrecDNA and followed by the polyA tail of the SV40. We found four mice with positive DNA genotype and three probable sites of genomic integration of the transgene. Only two of the founders showed presence of Cre-mRNA and production of Cre protein, restricted to megakaryocytes. The activity of Cre in mediating gene targeting was assessed by crossing mαIIb-cre mice to Cre-reporter mice (ROSA26-lacZ). The activity of β-galactosidase, detected only in megakaryocytes, was sufficient to generate intense staining of X-Gal in hepatic haematopoietic islands of 14.5 dpc fetuses, in bone marrow megakaryocytes and platelets from adult mice as well as in vitro cultured megakaryocytes differentiated from bone marrow hematopoietic stem cells. Moreover, the recombinase activity was sufficient to produce the specific gene targeting of a floxed CD40L allele in megakaryocytes. The mαIIb-cre transgenic mice with restricted production of Cre in megakaryocytes, offers a selective, alternative, new tool for the genetic analysis of platelet pathophysiology.

Expression of the glycoprotein hormone alpha-subunit gene in the placenta requires a functional cyclic AMP response element, whereas a different cis-acting element mediates pituitary-specific expression

1989 ◽  
Vol 9 (11) ◽  
pp. 5113-5122
Author(s):  
J A Bokar ◽  
R A Keri ◽  
T A Farmerie ◽  
R A Fenstermaker ◽  
B Andersen ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Transgenic Mice ◽  
Regulatory Region ◽  
Alpha Subunit ◽  
Subunit Gene ◽  
Glycoprotein Hormone ◽  
Specific Expression ◽  
Response Element ◽  
Cyclic Amp Response Element ◽  
Choriocarcinoma Cells

The single-copy gene encoding the alpha subunit of glycoprotein hormones is expressed in the pituitaries of all mammals and in the placentas of only primates and horses. We have systematically analyzed the promoter-regulatory elements of the human and bovine alpha-subunit genes to elucidate the molecular mechanisms underlying their divergent patterns of tissue-specific expression. This analysis entailed the use of transient expression assays in a chorionic gonadotropin-secreting human choriocarcinoma cell line, protein-DNA binding assays, and expression of chimeric forms of human or bovine alpha subunit genes in transgenic mice. From the results, we conclude that placental expression of the human alpha-subunit gene requires a functional cyclic AMP response element (CRE) that is present as a tandem repeat in the promoter-regulatory region. In contrast, the promoter-regulatory region of the bovine alpha-subunit gene, as well as of the rat and mouse genes, was found to contain a single CRE homolog that differed from its human counterpart by a single nucleotide. This difference substantially reduced the binding affinity of the bovine CRE homolog for the nuclear protein that bound to the human alpha CRE and thereby rendered the bovine alpha-subunit promoter inactive in human choriocarcinoma cells. However, conversion of the bovine alpha CRE homolog to an authentic alpha CRE restored activity to the bovine alpha-subunit promoter in choriocarcinoma cells. Similarly, a human but not a bovine alpha transgene was expressed in placenta in transgenic mice. Thus, placenta-specific expression of the human alpha-subunit gene may be the consequence of the recent evolution of a functional CRE. Expression of the human alpha transgene in mouse placenta further suggests that evolution of placenta-specific trans-acting factors preceded the appearance of this element. Finally, in contrast to their divergent patterns of placental expression, both the human and bovine alpha-subunit transgenes were expressed in mouse pituitary, indicating differences in the composition of the enhancers required for pituitary- and placenta-specific expression.

Localization of distal regulatory domains in the megakaryocyte-specific platelet basic protein/platelet factor 4 gene locus

Blood ◽  
2001 ◽  
Vol 98 (3) ◽  
pp. 610-617 ◽  
Author(s):  
Chunyan Zhang ◽  
Michael A. Thornton ◽  
M. Anna Kowalska ◽  
Bruce S. Sachis ◽  
Michael Feldman ◽  
...  
Keyword(s):  
Gene Locus ◽  
Basic Protein ◽  
Regulatory Region ◽  
Platelet Factor 4 ◽  
Specific Gene ◽  
Sufficient Information ◽  
Specific Expression ◽  
Platelet Factor ◽  
High Level

Abstract The genes for the related human (h) chemokines, PBP (platelet basic protein) and PF4 (platelet factor 4), are within 5.3 kilobases (kb) of each other and form a megakaryocyte-specific gene locus. The hypothesis was considered that the PBP and PF4 genes share a common distal regulatory region(s) that leads to their high-level megakaryocyte-specific expression in vivo. This study examined PBP and PF4 expression in transgenic mice using 4 distinct humanPBP/PF4 gene locus constructs. These studies showed that within the region studied there was sufficient information to regulate tissue-specific expression of both hPBP and hPF4. Indeed this region contained sufficient DNA information to lead to expression levels of PBP and PF4 comparable to the homologous mouse genes in a position-independent, copy number–dependent fashion. These studies also indicated that the DNA domains that led to this expression were distinct for the 2 genes; hPBP expression is regulated by a region that is 1.5 to 4.4 kb upstream of that gene. Expression of hPF4 is regulated by a region that is either intergenic between the 2 genes or immediately downstream of the hPF4 gene. Comparison of the available human and mouse sequences shows conserved flanking region domains containing potential megakaryocyte-related transcriptional factor DNA-binding sites. Further analysis of these regulatory regions may identify enhancer domains involved in megakaryopoiesis that may be useful in the selective expression of other genes in megakaryocytes and platelets as a strategy for regulating hemostasis, thrombosis, and inflammation.

Regulation of lineage-specific transcription of the sucrase-isomaltase gene in transgenic mice and cell lines

1995 ◽  
Vol 269 (6) ◽  
pp. G925-G939 ◽  
Author(s):  
A. J. Markowitz ◽  
G. D. Wu ◽  
A. Bader ◽  
Z. Cui ◽  
L. Chen ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cell Lines ◽  
Reporter Gene ◽  
Molecular Mechanisms ◽  
Ectopic Expression ◽  
Transgenic Animals ◽  
Intestinal Cell ◽  
Specific Gene ◽  
Specific Expression ◽  
Endogenous Gene

Sucrase-isomaltase (SI), a gene expressed exclusively in absorptive enterocytes, was used to examine the molecular mechanisms that regulate cell-specific gene expression in the intestinal epithelium. Transgenic mice were made with a construct containing nucleotides -8,500 to +54 of the mouse SI gene linked to a human growth hormone reporter gene. In adult transgenic animals, high-level transgene expression was limited to the small intestine, with low levels of ectopic expression in the colon. In contrast to the endogenous gene that is expressed only in enterocytes, the transgene was expressed in all four cell lineages, including enterocytes, enteroendocrine, goblet, and Paneth cells. To examine this process of lineage-specific expression further we studied Caco-2 and COLO DM cell lines, which model enterocytes and enteroendocrine cells, respectively. Reminiscent of results in transgenic animals, only Caco-2 cells transcribed the endogenous SI gene, whereas both Caco-2 and COLO DM cells supported transcription from chimeric SI reporter gene constructs. Taken together, these data suggest that each intestinal cell lineage has the cellular machinery to transcribe the SI gene. Moreover, these findings imply that transcription is normally repressed in nonenterocytic cells, possibly via a transcriptional silencer residing outside of the region of the SI gene examined in these studies.

scholarly journals A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9

2015 ◽  
Vol 113 (2) ◽  
pp. 338-343 ◽  
Author(s):  
Kelli J. Carroll ◽  
Catherine A. Makarewich ◽  
John McAnally ◽  
Douglas M. Anderson ◽  
Lorena Zentilin ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Gene Deletion ◽  
Disease Modeling ◽  
Embryonic Lethality ◽  
Null Alleles ◽  
Specific Gene ◽  
Specific Expression ◽  
Guide Rna ◽  
Cardiac Gene ◽  
Genomic Editing

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart.

A 286 bp upstream regulatory region of a rice anther-specific gene, OSIPP3, confers pollen-specific expression in Arabidopsis

2012 ◽  
Vol 35 (3) ◽  
pp. 455-462 ◽  
Author(s):  
Reema Khurana ◽  
Hitesh Kathuria ◽  
Arnab Mukhopadhyay ◽  
Sanjay Kapoor ◽  
Akhilesh K. Tyagi
Keyword(s):  
Regulatory Region ◽  
Specific Gene ◽  
Upstream Regulatory Region ◽  
Specific Expression

Identification of the sequences controlling cyclic AMP regulation and cell-type-specific expression of a prestalk-specific gene in Dictyostelium discoideum

1987 ◽  
Vol 7 (1) ◽  
pp. 149-159
Author(s):  
S Datta ◽  
R A Firtel
Keyword(s):  
Amino Acids ◽  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Base Pair ◽  
Regulatory Region ◽  
Specific Gene ◽  
Cell Type ◽  
Specific Expression ◽  
Endogenous Gene ◽  
Cell Type Specific Expression

We have cloned and analyzed a developmentally and spatially regulated prestalk cell-specific gene from Dictyostelium discoideum. The gene encodes a protein highly homologous to the lysosomal cysteine proteinases cathepsin H and cathepsin B. Amino acid comparisons between these enzymes showed that the active-site amino acids were conserved, as were amino acids known to be important for catalysis and residues which form the intramolecular cysteine bridges. We have constructed a series of internal deletions, duplications, and linker scanner mutations within the region 300 base pairs 5' to the cap site. Analysis of expression of the mutations in transformants identified a approximately 35-base pair GC-rich region containing a dAdC/dGdT palindromic repeat and a G-rich box which is homologous to the 3' GT half of the palindromic repeat. Deletion or disruption of the G box resulted in a approximately 50-fold drop in the level of expression of the gene fusion in transformants in response to cyclic AMP in single-cell culture but did not affect the temporal pattern of regulation or control by cyclic AMP. The expression of such constructs during normal multicellular differentiation paralleled that of the endogenous gene; however, the level of RNA from the constructs was only approximately 10-fold lower than that of constructs containing the G box. Deletion of the 3' half of the palindromic sequence and the G box region resulted in a dramatic decrease in the level of transcription, although the constructs still showed proper temporal expression. These results suggest that this 35-base-pair region acts as an important part of the regulatory region for cell type and cyclic AMP regulation.

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