Renal Anemia Model Mouse Established by Transgenic Rescue with an Erythropoietin Gene Lacking Kidney-Specific Regulatory Elements

ABSTRACT The erythropoietin (Epo) gene is under tissue-specific inducible regulation. Because the kidney is the primary EPO-producing tissue in adults, impaired EPO production in chronic kidney disorders results in serious renal anemia. The Epo gene contains a liver-specific enhancer in the 3′ region, but the kidney-specific enhancer for gene expression in renal EPO-producing (REP) cells remains elusive. Here, we examined a conserved upstream element for renal E po regulation (CURE) region that spans 17.4 kb to 3.6 kb upstream of the Epo gene and harbors several phylogenetically conserved elements. We prepared various Epo gene-reporter constructs utilizing a bacterial artificial chromosome and generated a number of transgenic-mouse lines. We observed that deletion of the CURE region (δCURE) abrogated Epo gene expression in REP cells. Although transgenic expression of the δCURE construct rescued Epo-deficient mice from embryonic lethality, the rescued mice had severe EPO-dependent anemia. These mouse lines serve as an elaborate model for the search for erythroid stimulatory activity and are referred to as AnRED (anemic model with renal EPO deficiency) mice. We also dissected the CURE region by exploiting a minigene harboring four phylogenetically conserved elements in reporter transgenic-mouse analyses. Our analyses revealed that Epo gene regulation in REP cells is a complex process that utilizes multiple regulatory influences.

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A regulatory region from the mouse Hox-2.2 promoter directs gene expression into developing limbs

Development ◽

10.1242/dev.112.3.807 ◽

1991 ◽

Vol 112 (3) ◽

pp. 807-811 ◽

Cited By ~ 5

Author(s):

K. Schughart ◽

C.J. Bieberich ◽

R. Eid ◽

F.H. Ruddle

Keyword(s):

Gene Expression ◽

Transgenic Mouse ◽

Reporter Gene ◽

Developmental Stages ◽

Regulatory Region ◽

Homeobox Gene ◽

Regulatory Elements ◽

Body Region ◽

Lacz Gene ◽

Mouse Lines

To characterize cis-acting regulatory elements of the murine homeobox gene, Hox-2.2, transgenic mouse lines were generated that contained the LacZ reporter gene under the control of different fragments from the presumptive Hox-2.2 promoter. A promoter region of 3600 base pairs (bp) was identified, which reproducibly directed reporter gene expression into specific regions of developing mouse embryos. At 8.5 days postcoitum (p.c.) reporter gene activity was detected in posterior regions of the lateral mesoderm and, in subsequent developmental stages, expression of the LacZ gene was restricted to specific regions of the developing limb buds and the mesenchyme of the ventrolateral body region. This pattern of Hox-2.2-LacZ expression was found in all transgenic embryos that have been generated with the 3.6 kb promoter fragment (two founder embryos and embryos from five transgenic lines). In addition, embryos from two transgenic mouse lines expressed the reporter gene at low levels in the developing central nervous system (CNS). Our results are consistent with the idea that in addition to their presumptive role in CNS and vertebrae development, Hox-2.2 gene products are involved in controlling pattern formation in developing limbs.

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Sources of off-target expression from recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915974116 ◽

2019 ◽

Vol 116 (52) ◽

pp. 27001-27010 ◽

Cited By ~ 7

Author(s):

Kyle B. Fischer ◽

Hannah K. Collins ◽

Edward M. Callaway

Keyword(s):

Gene Expression ◽

Transgenic Mouse ◽

Transgene Expression ◽

Cell Types ◽

Cell Type ◽

Recognition Sites ◽

Highly Sensitive ◽

Plasmid Production ◽

Targeted Gene Expression ◽

Mouse Lines

In combination with transgenic mouse lines expressing Cre or Flp recombinases in defined cell types, recombinase-dependent adeno-associated viruses (AAVs) have become the tool of choice for localized cell-type-targeted gene expression. Unfortunately, applications of this technique when expressing highly sensitive transgenes are impeded by off-target, or “leak” expression, from recombinase-dependent AAVs. We investigated this phenomenon and find that leak expression is mediated by both infrequent transcription from the inverted transgene in recombinant-dependent AAV designs and recombination events during bacterial AAV plasmid production. Recombination in bacteria is mediated by homology across the antiparallel recombinase-specific recognition sites present in recombinase-dependent designs. To address both of these issues we designed an AAV vector that uses mutant “cross-over insensitive” recognition sites combined with an “ATG-out” design. We show that these CIAO (cross-over insensitive ATG-out) vectors virtually eliminate leak expression. CIAO vectors provide reliable and targeted transgene expression and are extremely useful for recombinase-dependent expression of highly sensitive transgenes.

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−245 bp of 5′-Flanking Region From the Human Platelet Factor 4 Gene Is Sufficient to Drive Megakaryocyte-Specific Expression In Vivo

Blood ◽

10.1182/blood.v91.7.2326.2326_2326_2333 ◽

1998 ◽

Vol 91 (7) ◽

pp. 2326-2333 ◽

Cited By ~ 1

Author(s):

Zheng Cui ◽

Michael P. Reilly ◽

Saul Surrey ◽

Elias Schwartz ◽

Steven E. McKenzie

Keyword(s):

Gene Expression ◽

Transgenic Mouse ◽

Reporter Gene ◽

Human Platelet ◽

Platelet Factor 4 ◽

Reporter Gene Expression ◽

Platelet Factor ◽

Flanking Region ◽

Mouse Lines

Platelet factor 4 (PF4) serves as a lineage-specific marker of megakaryocyte development. We previously identified two positively acting sequences in the human platelet factor 4 (hPF4) gene promoter that synergized to drive high-level luciferase reporter gene expression in vitro. Using portions of the hPF4 5′-flanking region linked to the lacZ reporter gene, we observed in this investigation that constructs with −245 bp of 5′-flanking region were more active than constructs with −2 kb of 5′-flanking region in vitro. We created two independent transgenic mouse lines with a −245-bp hPF4/lacZ construct. Cells from these mice were tested for β-galactosidase (β-gal) expression at the mRNA level by Northern blot and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and at the protein level by immunohistochemistry assay. Mice from one line showed β-gal expression specifically in all megakaryocytes of all ploidy classes from bone marrow and in platelets. Expression level was comparable to that driven by the 1.1-kb rat PF4 promoter in other transgenic mouse lines. Those in the second line showed no β-gal expression in megakaryocytes, platelets, or any of the eight organs tested. The −245-bp hPF4 promoter is capable of driving reporter gene expression in a megakaryocyte-specific manner in transgenic mice. The small size of this megakaryocyte-specific promoter is compatible with that required in some viral vectors and may provide a model for targeting gene expression to megakaryocytes.

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Adenoviral nerve growth factor and β-galactosidase transfer to spinal cord: a behavioral and histological analysis

Journal of Neurosurgery Spine ◽

10.3171/spi.1999.90.1.0099 ◽

1999 ◽

Vol 90 (1) ◽

pp. 99-108 ◽

Cited By ~ 8

Author(s):

Nicholas M. Boulis ◽

Vikas Bhatia ◽

Theodore I. Brindle ◽

Harland T. Holman ◽

Daniel J. Krauss ◽

...

Keyword(s):

Gene Expression ◽

Spinal Cord ◽

Nerve Growth Factor ◽

Viral Gene Expression ◽

Adenoviral Vectors ◽

Viral Gene ◽

Content Type ◽

Transgenic Expression ◽

Spinal Cord Function ◽

Fine Print

Object. The present study characterizes the time course and loci of gene expression induced by the administration of adenoviral vectors into spinal cord. Although a marked inflammatory response to these vectors occurred, no effect on spinal cord function was seen in the 1st postoperative week. The expression of transgenic genes delivered by viral vectors is being exploited throughout the nervous system. The present study utilized adenoviral vectors containing the Rous sarcoma virus (RSV) promoter and a nuclear localization signal to achieve transgenic expression in mammalian spinal cord. Methods. Initial experiments utilizing the vector Ad.RSVlacZ (1012 particles/ml) injected into the region of the central canal resulted in viral gene expression stretching over approximately 1.2 cm of spinal cord. Gene expression was first detected 3 days following viral administration and lasted until postinjection Day 14 with peak expression at Day 7. A variety of cell types in both white and gray matter expressed lacZ. Transgenic expression of the neurotrophin nerve growth factor (NGF) was achieved using injections of Ad.RSVNGF. On histological examination mononuclear inflammatory infiltrate and gliosis were revealed surrounding the injection sites of spinal cords receiving adenovirus but not vehicle. To assess spinal cord function during viral gene expression, animals previously trained in an operant runway task were tested at 7 days postinjection (the peak of viral gene expression) and demonstrated no changes in spinal cord function. Conclusions. Results of this study using adenoviral neurotrophic gene transfer indicate that it provided an effective tool for the delivery of potentially therapeutic proteins to the injured or diseased spinal cord.

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−245 bp of 5′-Flanking Region From the Human Platelet Factor 4 Gene Is Sufficient to Drive Megakaryocyte-Specific Expression In Vivo

Blood ◽

10.1182/blood.v91.7.2326 ◽

1998 ◽

Vol 91 (7) ◽

pp. 2326-2333 ◽

Cited By ~ 9

Author(s):

Zheng Cui ◽

Michael P. Reilly ◽

Saul Surrey ◽

Elias Schwartz ◽

Steven E. McKenzie

Keyword(s):

Gene Expression ◽

Transgenic Mouse ◽

Reporter Gene ◽

Human Platelet ◽

Platelet Factor 4 ◽

Reporter Gene Expression ◽

Platelet Factor ◽

Flanking Region ◽

Mouse Lines

Abstract Platelet factor 4 (PF4) serves as a lineage-specific marker of megakaryocyte development. We previously identified two positively acting sequences in the human platelet factor 4 (hPF4) gene promoter that synergized to drive high-level luciferase reporter gene expression in vitro. Using portions of the hPF4 5′-flanking region linked to the lacZ reporter gene, we observed in this investigation that constructs with −245 bp of 5′-flanking region were more active than constructs with −2 kb of 5′-flanking region in vitro. We created two independent transgenic mouse lines with a −245-bp hPF4/lacZ construct. Cells from these mice were tested for β-galactosidase (β-gal) expression at the mRNA level by Northern blot and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and at the protein level by immunohistochemistry assay. Mice from one line showed β-gal expression specifically in all megakaryocytes of all ploidy classes from bone marrow and in platelets. Expression level was comparable to that driven by the 1.1-kb rat PF4 promoter in other transgenic mouse lines. Those in the second line showed no β-gal expression in megakaryocytes, platelets, or any of the eight organs tested. The −245-bp hPF4 promoter is capable of driving reporter gene expression in a megakaryocyte-specific manner in transgenic mice. The small size of this megakaryocyte-specific promoter is compatible with that required in some viral vectors and may provide a model for targeting gene expression to megakaryocytes.

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Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model

PLoS Biology ◽

10.1371/journal.pbio.3000749 ◽

2020 ◽

Vol 18 (11) ◽

pp. e3000749

Author(s):

Rui Li ◽

Xianyou Xia ◽

Xing Wang ◽

Xiaoyu Sun ◽

Zhongye Dai ◽

...

Keyword(s):

Gene Expression ◽

Mouse Model ◽

Ex Vivo ◽

Embryonic Stem ◽

Regulatory Elements ◽

Loss Of Function ◽

Transgenic Expression ◽

Guide Rnas ◽

High Throughput Screens

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 has been widely used far beyond genome editing. Fusions of deactivated Cas9 (dCas9) to transcription effectors enable interrogation of the epigenome and controlling of gene expression. However, the large transgene size of dCas9-fusion hinders its applications especially in somatic tissues. Here, we develop a robust CRISPR interference (CRISPRi) system by transgenic expression of doxycycline (Dox) inducible dCas9-KRAB in mouse embryonic stem cells (iKRAB ESC). After introduction of specific single-guide RNAs (sgRNAs), the induced dCas9-KRAB efficiently maintains gene inactivation, although it modestly down-regulates the expression of active genes. The proper timing of Dox addition during cell differentiation or reprogramming allows us to study or screen spatiotemporally activated promoters or enhancers and thereby the gene functions. Furthermore, taking the ESC for blastocyst injection, we generate an iKRAB knock-in (KI) mouse model that enables the shutdown of gene expression and loss-of-function (LOF) studies ex vivo and in vivo by a simple transduction of gRNAs. Thus, our inducible CRISPRi ESC line and KI mouse provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements in the setting of development or diseases.

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Bacterial Artificial Chromosome Mutagenesis Using Recombineering

Journal of Biomedicine and Biotechnology ◽

10.1155/2011/971296 ◽

2011 ◽

Vol 2011 ◽

pp. 1-10 ◽

Cited By ~ 21

Author(s):

Kumaran Narayanan ◽

Qingwen Chen

Keyword(s):

Gene Expression ◽

Bacterial Artificial Chromosome ◽

Restriction Enzymes ◽

Artificial Chromosome ◽

Regulatory Elements ◽

E Coli ◽

Functional Studies ◽

Large Size

Gene expression from bacterial artificial chromosome (BAC) clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development ofin vivohomologous recombination strategies based on recombineering inE. colihas helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACsin vitroandin vivo.

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Generation and Validation of Versatile Inducible CRISPRi Embryonic Stem Cell and Mouse Models

10.1101/2020.04.28.065854 ◽

2020 ◽

Author(s):

Rui Li ◽

Xianyou Xia ◽

Xing Wang ◽

Xiaoyu Sun ◽

Zhongye Dai ◽

...

Keyword(s):

Gene Expression ◽

Ex Vivo ◽

Embryonic Stem ◽

Regulatory Elements ◽

Loss Of Function ◽

Transgenic Expression ◽

Somatic Tissues ◽

Gene Functions ◽

High Throughput Screens

ABSTRACTCRISPR-Cas9 has been widely used far beyond genome editing. Fusions of deactivated Cas9 (dCas9) to transcription effectors enable interrogation of the epigenome and controlling of gene expression. However the large transgene size of dCas9-fusion hinders its applications especially in somatic tissues. Here, we develop a robust CRISPR interference (CRISPRi) system by transgenic expression of doxycycline (Dox) inducible dCas9-KRAB in mouse embryonic stem cells (iKRAB ESC). After introduction of specific gRNAs, the induced dCas9-KRAB efficiently maintains gene inactivation, though it exerts modest effects on active gene expression. Proper timing of Dox addition during cell differentiation or reprogramming allows us to study or screen spatiotemporally activated promoters or enhancers and thereby the gene functions. Furthermore, taking the ESC for blastocyst injection, we generate an iKRAB knockin (KI) mouse model that enables shut-down of gene expression and loss-of-function studies ex vivo and in vivo by a simple transduction of gRNAs. Thus, our inducible CRISPRi ESC line and KI mouse provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements in the setting of development or diseases.

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Promoter Boundaries for theluxCDABEandbetIBA-proXWVOperons inVibrio harveyiDefined by the Method Rapid Arbitrary PCR Insertion Libraries (RAIL)

Journal of Bacteriology ◽

10.1128/jb.00724-17 ◽

2018 ◽

Vol 200 (11) ◽

Cited By ~ 2

Author(s):

Christine M. Hustmyer ◽

Chelsea A. Simpson ◽

Stephen G. Olney ◽

Douglas B. Rusch ◽

Matthew L. Bochman ◽

...

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Transcriptional Regulation ◽

Vibrio Harveyi ◽

Reporter Genes ◽

Regulatory Elements ◽

Model Systems ◽

New Method ◽

Regulatory Sequences ◽

Content Type

ABSTRACTExperimental studies of transcriptional regulation in bacteria require the ability to precisely measure changes in gene expression, often accomplished through the use of reporter genes. However, the boundaries of promoter sequences required for transcription are often unknown, thus complicating the construction of reporters and genetic analysis of transcriptional regulation. Here, we analyze reporter libraries to define the promoter boundaries of theluxCDABEbioluminescence operon and thebetIBA-proXWVosmotic stress operon inVibrio harveyi. We describe a new method calledrapidarbitrary PCRinsertionlibraries (RAIL) that combines the power of arbitrary PCR and isothermal DNA assembly to rapidly clone promoter fragments of various lengths upstream of reporter genes to generate large libraries. To demonstrate the versatility and efficiency of RAIL, we analyzed the promoters driving expression of theluxCDABEandbetIBA-proXWVoperons and created libraries of DNA fragments from these loci fused to fluorescent reporters. Using flow cytometry sorting and deep sequencing, we identified the DNA regions necessary and sufficient for maximum gene expression for each promoter. These analyses uncovered previously unknown regulatory sequences and validated known transcription factor binding sites. We applied this high-throughput method togfp,mCherry, andlacZreporters and multiple promoters inV. harveyi. We anticipate that the RAIL method will be easily applicable to other model systems for genetic, molecular, and cell biological applications.IMPORTANCEGene reporter constructs have long been essential tools for studying gene regulation in bacteria, particularly following the recent advent of fluorescent gene reporters. We developed a new method that enables efficient construction of promoter fusions to reporter genes to study gene regulation. We demonstrate the versatility of this technique in the model bacteriumVibrio harveyiby constructing promoter libraries for three bacterial promoters using three reporter genes. These libraries can be used to determine the DNA sequences required for gene expression, revealing regulatory elements in promoters. This method is applicable to various model systems and reporter genes for assaying gene expression.

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Putative intestine-specific enhancers located in 5′ sequence of the CDX1 gene regulate CDX1 expression in the intestine

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00326.2003 ◽

2004 ◽

Vol 286 (5) ◽

pp. G872-G880 ◽

Cited By ~ 6

Author(s):

Erinn B. Rankin ◽

Wei Xu ◽

Debra G. Silberg ◽

EunRan Suh

Keyword(s):

Gene Expression ◽

Intestinal Epithelium ◽

Critical Role ◽

Ectopic Expression ◽

Regulatory Elements ◽

Intestinal Epithelial ◽

Transgenic Expression ◽

Homeobox Transcription Factor ◽

Flanking Sequences ◽

Cell Growth And Differentiation

CDX1 is a homeobox transcription factor that plays a critical role in intestinal epithelial cell growth and differentiation. CDX1 gene expression is tightly regulated in a temporal and cell-type specific manner. However, very little is known about the regulatory mechanisms that direct CDX1 gene expression in the intestine. To elucidate these mechanisms, we employed a series of transgenic mouse studies using the 5′ flanking sequences of the human CDX1 gene. Transgenic mice containing nucleotides between -5667 and +68 relative to the transcription start site of the CDX1 gene demonstrated ectopic expression of the transgene in the brain and gastric smooth muscle. However, transgenic expression of the nucleotides -15601 to +68 of the CDX1 gene was restricted to the intestinal epithelium, which was identical to endogenous CDX1 gene expression. Taken together, the upstream sequences between -15601 and -5667 contain regulatory elements that direct transgene expression specifically to the intestinal epithelium. Furthermore, DNase I hypersensitivity assays revealed two active chromatin regions in the CDX1 gene (hypertensive sites 1 and 2) located at approximately -5.8 and -6.8 kb upstream of the CDX1 gene, respectively, which may function as potential intestine-specific enhancers.

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