scholarly journals The 5′ flanking region of the rat LAP (C/EBPβ) gene can direct high-level, position-independent, copy number dependent expression in multiple tissues in transgenic mice

1994 ◽  
Vol 22 (5) ◽  
pp. 756-766 ◽  
Author(s):  
Dale Talbot ◽  
Patrick Descombes ◽  
Ueli Schibler
Keyword(s):  
Transgenic Mice ◽  
Copy Number ◽  
Independent Copy ◽  
Flanking Region ◽  
High Level

The Human Myeloperoxidase Promoter Directs Hematopoietic Cell-Specific Transcription in Transgenic Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4202-4202
Author(s):  
Congjun Yao ◽  
William D. Martin ◽  
Kimberly Works ◽  
Garth Austin
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Tissue Specificity ◽  
Luciferase Activity ◽  
Renilla Luciferase ◽  
Promoter Construct ◽  
Flanking Region ◽  
High Level ◽  
Renilla Luciferase Activity ◽  
Mouse Lines

Abstract The myeloperoxidase (MPO) gene is expressed specifically in immature myeloid cells and not to a detectable extent in other cell types or in more mature myeloid cells. Furthermore, the MPO gene is actively transcribed both in normal myeloblasts (MPO protein constitutes about 5% of total protein in granulocytes) and in the myeloblasts of the majority of acute myeloid leukemias, but not in lymphoid leukemias or non-myeloid tumors. This has led to use of MPO activity for many years as the defining cytochemical marker for myeloid differentiation in anatomic pathology. We were the first to purify the human MPO promoter and to dissect many of its regulatory elements. Later, we showed the existence of three distinct human MPO promoters. More recently, we demonstrated that only one of these, which we term “P1”, is responsible for most of the physiologic MPO transcription of the human MPO gene. While until recently, purified human (or murine) MPO promoter constructs have exhibited only partial tissue specificity, we recently obtained a human MPO promoter construct whose specificity in cell culture mimics that of MPO gene expression seen in vivo. Comparison of many different promoter constructs reveals optimal, myeloid-specific activity for a construct extending from bp −4193 in the 5′-flanking region of the MPO gene to bp +15, just downstream from the transcription start site. Inclusion of additional downstream sequences did not increase promoter activity or specificity. We have now developed transgenic murine lines in which 4.2 kb of human MPO proximal 5′ flanking region DNA was linked to an adjacent downstream Renilla luciferase reporter (MPORLUC). A series of founder mouse lines were screened by PCR assays of DNA extracted from the tail vein for the presence of the MPO promoter-Renilla luciferase construct. Five founder mouse lines were positive for the transgene by the PCR assay. Transgenic and control mice (founders or offspring) were sacrificed and all possible tissues were examined for Renilla luciferase activity by an enzymatic assay (Promega). High-level Renilla luciferase activity was observed in two founder lines but only one of these founders produced offspring. In each of multiple different mice belonging to the founder line which was successfully mated, high-level Renilla luciferase activity was evident in bone marrow of femur and to a variable degree in vertebral bone marrow. In mice younger than four months old, a modest-level of luciferase activity was also observed in spleen. Luciferase activity was at or near background levels in thymus, heart, lung, liver, kidney, stomach, colon, bladder, brain, skeletal muscle, skin and small intestine in all of the MPORLUC transgenic mice. All tissues in wild type mice lacked measurable Renilla luciferase activity. Interestingly, there has been a slightly higher level of deaths in the luciferase-positive transgenic mice than in controls, suggesting an as yet uncharacterized toxic effect of the transgene. These results indicate that the human MPO promoter construct used in these studies exhibits tissue specificity of activity. Such a promoter might be useful for future gene targeting studies or for gene therapy.

A minimal c-fes cassette directs myeloid-specific expression in transgenic mice

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3040-3048
Author(s):  
Ahlke Heydemann ◽  
Soren Warming ◽  
Cynthia Clendenin ◽  
Kirsten Sigrist ◽  
J. Peter Hjorth ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Dna Sequences ◽  
Copy Number ◽  
Messenger Rna ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Control Function ◽  
Integration Site ◽  
Myeloid Cells ◽  
Independent Copy

The c-fes proto-oncogene encodes a 92-kd protein tyrosine kinase whose expression is restricted largely to myeloid and endothelial cells in adult mammals. A 13.2-kilobase (kb) humanc-fes genomic fragment was previously shown to containcis-acting element(s) sufficient for a locus control function in bone marrow macrophages. Locus control regions (LCRs) confer transgene expression in mice that is integration site independent, copy number dependent, and similar to endogenous murine messenger RNA levels. To identify sequences required for this LCR,c-fes transgenes were analyzed in mice. Myeloid-cell–specific, deoxyribonuclease-I–hypersensitive sites localized to the 3′ boundary of exon 1 and intron 3 are required to confer high-level transgene expression comparable to endogenous c-fes, independent of integration site. We define a minimal LCR element as DNA sequences (nucleotides +28 to +2523 relative to the transcription start site) located within intron 1 to intron 3 of the human locus. When this 2.5-kb DNA fragment was linked to a c-fes complementary DNA regulated by its own 446–base-pair promoter, integration-site–independent, copy-number–dependent transcription was observed in myeloid cells in transgenic mice. Furthermore, this 2.5-kb cassette directed expression of a heterologous gene (enhanced green fluorescent protein) exclusively in myeloid cells. The c-fes regulatory unit represents a novel reagent for targeting gene expression to macrophages and neutrophils in transgenic mice.

A minimal c-fes cassette directs myeloid-specific expression in transgenic mice

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3040-3048 ◽  
Author(s):  
Ahlke Heydemann ◽  
Soren Warming ◽  
Cynthia Clendenin ◽  
Kirsten Sigrist ◽  
J. Peter Hjorth ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Dna Sequences ◽  
Copy Number ◽  
Messenger Rna ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Control Function ◽  
Integration Site ◽  
Myeloid Cells ◽  
Independent Copy

Abstract The c-fes proto-oncogene encodes a 92-kd protein tyrosine kinase whose expression is restricted largely to myeloid and endothelial cells in adult mammals. A 13.2-kilobase (kb) humanc-fes genomic fragment was previously shown to containcis-acting element(s) sufficient for a locus control function in bone marrow macrophages. Locus control regions (LCRs) confer transgene expression in mice that is integration site independent, copy number dependent, and similar to endogenous murine messenger RNA levels. To identify sequences required for this LCR,c-fes transgenes were analyzed in mice. Myeloid-cell–specific, deoxyribonuclease-I–hypersensitive sites localized to the 3′ boundary of exon 1 and intron 3 are required to confer high-level transgene expression comparable to endogenous c-fes, independent of integration site. We define a minimal LCR element as DNA sequences (nucleotides +28 to +2523 relative to the transcription start site) located within intron 1 to intron 3 of the human locus. When this 2.5-kb DNA fragment was linked to a c-fes complementary DNA regulated by its own 446–base-pair promoter, integration-site–independent, copy-number–dependent transcription was observed in myeloid cells in transgenic mice. Furthermore, this 2.5-kb cassette directed expression of a heterologous gene (enhanced green fluorescent protein) exclusively in myeloid cells. The c-fes regulatory unit represents a novel reagent for targeting gene expression to macrophages and neutrophils in transgenic mice.

Expression of SV40 T antigen under control of rabbit uteroglobin promoter in transgenic mice

1991 ◽  
Vol 261 (2) ◽  
pp. L70-L76 ◽  
Author(s):  
F. J. DeMayo ◽  
M. J. Finegold ◽  
T. N. Hansen ◽  
L. A. Stanley ◽  
B. Smith ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Fusion Gene ◽  
Female Offspring ◽  
T Antigen ◽  
Northern Analysis ◽  
Antigen Gene ◽  
Submucosal Glands ◽  
Flanking Region ◽  
Rabbit Uteroglobin ◽  
High Level

The rabbit uteroglobin gene is expressed in the lungs and reproductive tracts of male and female rabbits. To examine whether the promoter region of the uteroglobin gene could be used to target a heterologous gene to the lungs of transgenic mice, a fusion gene consisting of 3.3 kb of the 5'-flanking region of the rabbit uteroglobin gene and the large T antigen gene of the SV40 virus was constructed and microinjected into the pronuclei of one-cell mouse embryos. Eleven founder transgenic mice (5 female and 6 male) were generated. Seven of these mice developed bronchioalveolar neoplasms. Four of the founder males also developed primitive undifferentiated urogenital tract tumors. One founder female and one female offspring of a founder male developed glandular paraovarian tumors. Northern analysis revealed that the predominant site of expression of the transgene was the lung. Immunohistochemical staining showed T antigen predominantly in epithelial cells lining the bronchioles, the submucosal glands of the trachea, and the neoplasms. There appeared to be a high level of mosaicism for the transgene in the founder mice, with poor transmission of the transgene to subsequent generations. This suggests that, under the control of the uteroglobin promoter, the T antigen gene may be lethal to the fetus.

scholarly journals Analysis of the human alpha-globin gene cluster in transgenic mice

1993 ◽  
Vol 90 (23) ◽  
pp. 11262-11266 ◽  
Author(s):  
J A Sharpe ◽  
D J Wells ◽  
E Whitelaw ◽  
P Vyas ◽  
D R Higgs ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Gene Cluster ◽  
Copy Number ◽  
Globin Gene ◽  
Globin Genes ◽  
Regulation Of Expression ◽  
Globin Gene Cluster ◽  
Hypersensitive Sites ◽  
Alpha Globin ◽  
High Level

A 350-bp segment of DNA associated with an erythroid-specific DNase I-hypersensitive site (HS-40), upstream of the alpha-globin gene cluster, has been identified as the major tissue-specific regulator of the alpha-globin genes. However, this element does not direct copy number-dependent or developmentally stable expression of the human genes in transgenic mice. To determine whether additional upstream hypersensitive sites could provide more complete regulation of alpha gene expression we have studied 17 lines of transgenic mice bearing various DNA fragments containing HSs -33, -10, -8, and -4, in addition to HS -40. Position-independent, high-level expression of the human zeta- and alpha-globin genes was consistently observed in embryonic erythroid cells. However, the additional HSs did not confer copy-number dependence, alter the level of expression, or prevent the variable down-regulation of expression in adults. These results suggest that the region upstream of the human alpha-globin genes is not equivalent to that upstream of the beta locus and that although the two clusters are coordinately expressed, there may be differences in their regulation.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mice

1993 ◽  
Vol 13 (9) ◽  
pp. 5266-5275
Author(s):  
R D Palmiter ◽  
E P Sandgren ◽  
D M Koeller ◽  
R L Brinster
Keyword(s):  
Transgenic Mice ◽  
Regulatory Elements ◽  
High Level Expression ◽  
Hypersensitive Sites ◽  
Enhanced Expression ◽  
Rat Growth ◽  
Flanking Sequences ◽  
High Level ◽  
Distal Regulatory Elements ◽  
Flanking Regions

DNA regions of 10 and 7 kb that flank the mouse metallothionein II (MT-II) and MT-I genes, respectively, were combined with a minimally marked MT-I (MT-I*) gene and tested in transgenic mice. This construct resulted in (i) position-independent expression of MT-I* mRNA and copy number-dependent expression, (ii) levels of hepatic MT-I mRNA per cell per transgene that were about half that derived from endogenous MT-I genes, (iii) appropriate regulation by metals and hormones, and (iv) tissue distribution of transgene mRNA that resembled that of endogenous MT-I mRNA. These features were not observed when MT-I* was tested without the flanking regions. These MT-I flanking sequences also improved the expression of rat growth hormone reporter genes, with or without introns, that were under the control of the MT-I promoter. Moreover, they enhanced expression from two of four heterologous promoters/enhancers that were tested. Deletion analysis indicated that regions known to have DNase I-hypersensitive sites were necessary but not sufficient for high-level expression. These data suggest that the DNA regions flanking the mouse MT-I and MT-II genes have functions like the locus control regions described for other genes.

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