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]

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.

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Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3978-3990.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3978-3990

Author(s):

B Liu ◽

G D Hammer ◽

M Rubinstein ◽

M Mortrud ◽

M J Low

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Reporter Gene ◽

Transgene Expression ◽

Intermediate Lobe ◽

Mobility Shift ◽

Specific Expression ◽

Dna Elements ◽

Pomc Gene

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

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Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3978 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3978-3990 ◽

Cited By ~ 41

Author(s):

B Liu ◽

G D Hammer ◽

M Rubinstein ◽

M Mortrud ◽

M J Low

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Reporter Gene ◽

Transgene Expression ◽

Intermediate Lobe ◽

Mobility Shift ◽

Specific Expression ◽

Dna Elements ◽

Pomc Gene

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

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Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

Molecular and Cellular Biology ◽

10.1128/mcb.16.7.3245 ◽

1996 ◽

Vol 16 (7) ◽

pp. 3245-3254 ◽

Cited By ~ 31

Author(s):

V Ngô ◽

D Gourdji ◽

J N Laverrière

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Cell Lines ◽

Anterior Pituitary ◽

Regulatory Elements ◽

Specific Expression ◽

Site Specific ◽

Cpg Dinucleotides ◽

Cpg Sites

The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo.

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Negative regulation in correct tissue-specific expression of mouse mammary tumor virus in transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.5822 ◽

1990 ◽

Vol 10 (11) ◽

pp. 5822-5829 ◽

Cited By ~ 36

Author(s):

S R Ross ◽

C L Hsu ◽

Y Choi ◽

E Mok ◽

J P Dudley

Keyword(s):

Transgenic Mice ◽

Mouse Mammary Tumor Virus ◽

Reporter Genes ◽

Regulatory Elements ◽

Specific Expression ◽

Mammary Tumor Virus ◽

Mouse Mammary Tumor ◽

Tumor Virus ◽

Mmtv Ltr ◽

Inappropriate Expression

Mouse mammary tumor virus (MMTV) is an endogenous murine retrovirus that is expressed in the epithelial cells of the mammary and salivary glands, lungs, kidneys, and seminal vesicles and in the lymphoid cells of the spleen and thymus. Several studies have shown that the long terminal repeat (LTR) of this virus can direct the expression of reporter genes to the same tissues in transgenic mice. To determine whether multiple regulatory elements within the LTR are involved in this tissue-specific expression, we have established lines of transgenic mice containing transgenes that have deletions in the MMTV LTR. Deletions of all LTR sequences upstream of -364 or of LTR sequences from -165 to -665 both result in the expression of linked reporter genes such as the simian virus 40 early region or the bacterial enzyme chloramphenicol acetyltransferase in novel sites, such as the heart, brain, and skeletal muscle; expression of endogenous MMTV and transgenes containing the full-length LTR is not detected in these organs. Negative regulation appears to involve more than one region, since deletion of sequences between either -201 and -471 or -201 and -344, as well as sequences upstream of -364, results in inappropriate expression in heart, brain, and skeletal muscle. Therefore, a negative regulatory element(s) in the MMTV LTR can suppress transcription from the viral promoter in several different organs. This represents the first example of generalized negative regulatory elements that act in many different tissues in transgenic mice to prevent inappropriate expression of a gene.

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Leukocyte integrin CD11b promoter directs expression in lymphocytes and granulocytes in transgenic mice

Blood ◽

10.1182/blood.v85.4.1017.bloodjournal8541017 ◽

1995 ◽

Vol 85 (4) ◽

pp. 1017-1024 ◽

Cited By ~ 17

Author(s):

A Back ◽

K East ◽

D Hickstein

Keyword(s):

Transgenic Mice ◽

Reporter Gene ◽

Transgene Expression ◽

Human Leukocyte ◽

Surface Expression ◽

Regulatory Elements ◽

Specific Pattern ◽

Integrin Subunit ◽

Cd11b Expression

The human leukocyte integrin subunit CD11b is expressed predominantly on myelomonocytic cells. To identify CD11b promoter sequences important for myelomonocytic gene expression and to assess the utility of the CD11b promoter for expressing heterologous genes in vivo, we generated transgenic mice with a human CD4 reporter gene driven by CD11b promoter constructs composed of 1.5, 0.3, or 0.1 kb of DNA sequence 5′ to the transcription start site. Using flow cytometry to detect the human CD4 reporter on murine leukocytes, two of three 1.5-kb CD11b promoter founder lines showed surface expression of the human CD4 transgene in granulocytes and lymphocytes. The transgene expression observed in lymphocytes was inappropriate relative to the normal pattern of CD11b expression. Of the eight 0.3-kb or 0.1-kb founder lines, only one 0.1- kb founder line showed transgene expression. The overall pattern of transgene expression among the 11 founder lines does not parallel expression of the endogenous CD11b gene. These studies indicate that additional CD11b regulatory elements will be required to express a reporter gene in vivo in a lineage-specific pattern that mimics the endogenous CD11b gene.

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Ksp-cadherin gene promoter. II. Kidney-specific activity in transgenic mice

AJP Renal Physiology ◽

10.1152/ajprenal.1999.277.4.f599 ◽

1999 ◽

Vol 277 (4) ◽

pp. F599-F610 ◽

Cited By ~ 22

Author(s):

Peter Igarashi ◽

Cooduvalli S. Shashikant ◽

R. Brent Thomson ◽

Dilys A. Whyte ◽

Shuxian Liu-Chen ◽

...

Keyword(s):

Epithelial Cells ◽

Transgenic Mice ◽

Collecting Duct ◽

Gene Promoter ◽

Regulatory Elements ◽

Developmental Expression ◽

Tubular Epithelial Cells ◽

Specific Expression ◽

Tissue Specific

Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a tissue-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of tubular epithelial cells in the kidney. To determine the basis for tissue-specific expression of Ksp-cadherin in vivo, we evaluated the activity of the promoter in transgenic mice. Transgenic mice containing 3.3 kb of the mouse Ksp-cadherin promoter and an Escherichia coli lacZ reporter gene were generated by pronuclear microinjection. Assays of β-galactosidase enzyme activity showed that the transgene was expressed exclusively in the kidney in both adult and developing mice. Within the kidney, the transgene was expressed in a subset of renal tubular epithelial cells that endogenously expressed Ksp-cadherin and that were identified as collecting ducts by colabeling with Dolichos biflorus agglutinin. In the developing metanephros, expression of the transgene in the branching ureteric bud correlated with the developmental expression of Ksp-cadherin. Identical patterns of expression were observed in multiple founder mice, indicating that kidney specificity was independent of transgene integration site. However, heterocellular expression was observed consistent with repeat-induced gene silencing. We conclude that the Ksp-cadherin gene promoter directs kidney-specific expression in vivo. Regulatory elements that are sufficient to recapitulate the tissue- and differentiation-specific expression of Ksp-cadherin in the renal collecting duct are located within 3.3 kb upstream to the transcriptional start site.

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Negative regulation in correct tissue-specific expression of mouse mammary tumor virus in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.5822-5829.1990 ◽

1990 ◽

Vol 10 (11) ◽

pp. 5822-5829

Author(s):

S R Ross ◽

C L Hsu ◽

Y Choi ◽

E Mok ◽

J P Dudley

Keyword(s):

Transgenic Mice ◽

Mouse Mammary Tumor Virus ◽

Reporter Genes ◽

Regulatory Elements ◽

Specific Expression ◽

Mammary Tumor Virus ◽

Mouse Mammary Tumor ◽

Tumor Virus ◽

Mmtv Ltr ◽

Inappropriate Expression

Mouse mammary tumor virus (MMTV) is an endogenous murine retrovirus that is expressed in the epithelial cells of the mammary and salivary glands, lungs, kidneys, and seminal vesicles and in the lymphoid cells of the spleen and thymus. Several studies have shown that the long terminal repeat (LTR) of this virus can direct the expression of reporter genes to the same tissues in transgenic mice. To determine whether multiple regulatory elements within the LTR are involved in this tissue-specific expression, we have established lines of transgenic mice containing transgenes that have deletions in the MMTV LTR. Deletions of all LTR sequences upstream of -364 or of LTR sequences from -165 to -665 both result in the expression of linked reporter genes such as the simian virus 40 early region or the bacterial enzyme chloramphenicol acetyltransferase in novel sites, such as the heart, brain, and skeletal muscle; expression of endogenous MMTV and transgenes containing the full-length LTR is not detected in these organs. Negative regulation appears to involve more than one region, since deletion of sequences between either -201 and -471 or -201 and -344, as well as sequences upstream of -364, results in inappropriate expression in heart, brain, and skeletal muscle. Therefore, a negative regulatory element(s) in the MMTV LTR can suppress transcription from the viral promoter in several different organs. This represents the first example of generalized negative regulatory elements that act in many different tissues in transgenic mice to prevent inappropriate expression of a gene.

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Positive regulatory elements (HF-1a and HF-1b) and a novel negative regulatory element (HF-3) mediate ventricular muscle-specific expression of myosin light-chain 2-luciferase fusion genes in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1220-1229.1994 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1220-1229

Author(s):

K J Lee ◽

R Hickey ◽

H Zhu ◽

K R Chien

Keyword(s):

Transgenic Mice ◽

Myosin Light Chain ◽

Regulatory Element ◽

Regulatory Elements ◽

Luciferase Reporter ◽

Specific Expression ◽

Negative Regulatory Element ◽

Reporter Activity ◽

E Box

The cardiac myosin light-chain 2v (MLC-2v) gene has served as a model system to identify the pathways which restrict the expression of cardiac muscle genes to particular chambers of the heart during cardiogenesis. To identify the critical cis regulatory elements which mediate ventricular chamber-specific expression of the MLC-2v gene in the in vivo context, a series of transgenic mice which harbor mutations in putative MLC-2 cis regulatory elements in a 250-bp MLC-2-luciferase fusion gene which is expressed in a ventricular chamber-specific fashion in transgenic mice were generated. These studies demonstrate that both components of HF-1 (HF-1a and HF-1b/MEF-2) are required to maintain ventricular chamber-specific expression and function as positive regulatory elements. Mutations in another conserved element (HF-2) are without statistically significant effect on ventricular chamber expression. Transgenics harboring mutations in the E-box site also displayed significant upregulation of reporter activity in the soleus, gastrocnemius, and uterus, with a borderline effect on expression in liver. Mutations in another conserved element (HF-3) result in a marked (> 75-fold) upregulation of the luciferase reporter activity in the soleus muscle of multiple independent or transgenic founders. Since the HF-3 mutations appeared to have only a marginal effect on luciferase reporter activity in liver tissue, HF-3 appears to function as a novel negative regulatory element to primarily suppress expression in muscle tissues. Thus, a combination of positive (HF-1a/HF-1b) and negative (E-box and HF-3) regulatory elements appear to be required to maintain ventricular chamber-specific expression in the in vivo context.

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A modified pod specific promoter for high level heterologous expression of genes in legumes

Legume Research - An International Journal ◽

10.18805/lr-4073 ◽

2019 ◽

Author(s):

Aravind Kumar Konda ◽

Pallavi Singh ◽

Khela Ram Soren ◽

Narendra Pratap Singh

Keyword(s):

Gene Expression ◽

Regulatory Elements ◽

Specific Gene ◽

Specific Expression ◽

Tissue Specific ◽

Cis Acting ◽

Inducible Promoters ◽

Expression Of Genes ◽

Enhanced Expression ◽

High Level

Promoters are cis-acting regulatory elements that are usually present upstream to the coding sequences and determine the gene expression. Deployment of tissue specific and inducible promoters are constantly increasing for development of successful and stable multiple transgenic plants. To this end, as a strategy for enhanced expression of cis or transgenes, promoter engineering of the native msg promoter from soya bean has been carried out for executing pod specific expression of genes. Cis regulatory elements such as 5’UTR and poly (A) tract have been incorporated for imparting mRNA stability and translational enhancement to generate the modified 1.285 Kb pod specific promoter. Further to attain transcriptional enhancement the modified promoter has been cloned to generate Bi-directional Duplex Promoters (BDDP). The engineered msg promoter gene constructs can be deployed for high level tissue specific gene expression of cis/trans genes along with chosen terminator in chickpea. soybean and other legumes as well.

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