scholarly journals Spatial and temporal control of expression with light-gated LOV-LexA

2021 ◽  
Author(s):  
Inês MA Ribeiro ◽  
Wolfgang Eßbauer ◽  
Romina Kutlesa ◽  
Alexander Borst
Keyword(s):  
Single Neuron ◽  
Optic Lobe ◽  
Fat Body ◽  
Expression System ◽  
Cell Types ◽  
Temporal Control ◽  
Exogenous Genes ◽  
Ability To Drive ◽  
Isolated Cell ◽  
Control Of Expression

The ability to drive expression of exogenous genes in different tissues and cell types, under control of specific enhancers, has catapulted discovery in biology. While many enhancers drive expression broadly, several genetic tricks have been developed to obtain access to isolated cell types. However, studies of topographically organized neuropiles, such as the optic lobe in fruit flies, have raised the need for a system that can access subsets of cells within a single neuron type, a feat currently dependent on stochastic flip-out methods. To access the same subsets of cells consistently across flies, we developed LOV-LexA, a light-gated expression system based on the bacterial LexA transcription factor and the plant-derived LOV photosensitive domain. Expression of LOV-Lex in larval fat body as well as pupal and adult neurons enables spatial and temporal control of expression of transgenes under LexAop sequences with blue light. The LOV-LexA tool thus provides another layer of intersectional genetics, allowing for light-controlled genetic access to the same subsets of cells within an expression pattern across individual flies.

The function of the neurogenic genes during epithelial development in the Drosophila embryo

Development ◽  
1992 ◽  
Vol 116 (4) ◽  
pp. 1203-1220 ◽  
Author(s):  
A.Y. Hartenstein ◽  
A. Rugendorff ◽  
U. Tepass ◽  
V. Hartenstein
Keyword(s):  
Fat Body ◽  
Cell Types ◽  
Malpighian Tubules ◽  
Drosophila Embryo ◽  
Stomatogastric Nervous System ◽  
Mesodermal Cell ◽  
Epithelial Phenotype ◽  
In The Wild ◽  
Two Stages ◽  
Enhancer Of Split

The complex embryonic phenotype of the six neurogenic mutations Notch, mastermind, big brain, Delta, Enhancer of split and neuralized was analyzed by using different antibodies and PlacZ markers, which allowed us to label most of the known embryonic tissues. Our results demonstrate that all of the neurogenic mutants show abnormalities in many different organs derived from all three germ layers. Defects caused by the neurogenic mutations in ectodermally derived tissues fell into two categories. First, all cell types that delaminate from the ectoderm (neuroblasts, sensory neurons, peripheral glia cells and oenocytes) are increased in number. Secondly, ectodermal tissues that in the wild type form epithelial structures lose their epithelial phenotype and dissociate (optic lobe, stomatogastric nervous system) or show significant differentiative abnormalities (trachea, Malpighian tubules and salivary gland). Abnormalities in tissues derived from the mesoderm were observed in all six neurogenic mutations. Most importantly, somatic myoblasts do not fuse and/or form an aberrant muscle pattern. Cardioblasts (which form the embryonic heart) are increased in number and show differentiative abnormalities; other mesodermal cell types (fat body, pericardial cells) are significantly decreased. The development of the endoderm (midgut rudiments) is disrupted in most of the neurogenic mutations (Notch, Delta, Enhancer of split and neuralized) during at least two stages. Defects occur as early as during gastrulation when the invaginating midgut rudiments prematurely lose their epithelial characteristics. Later, the transition of the midgut rudiments to form the midgut epithelium does not occur. In addition, the number of adult midgut precursor cells that segregate from the midgut rudiments is strongly increased. We propose that, at least in the ectodermally and endodermally derived tissues, neurogenic gene function is primarily involved in interactions among cells that need to acquire or to maintain an epithelial phenotype.

Spatial and temporal control of expression of therapeutic genes using heat shock protein promoters

Methods ◽  
2005 ◽  
Vol 35 (2) ◽  
pp. 188-198 ◽  
Author(s):  
Claire Rome ◽  
Franck Couillaud ◽  
Chrit T.W. Moonen
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Temporal Control ◽  
Control Of Expression ◽  
Therapeutic Genes

scholarly journals Construction and Molecular Analysis of Gene Transfer Systems Derived from Bovine Immunodeficiency Virus

2001 ◽  
Vol 75 (7) ◽  
pp. 3371-3382 ◽  
Author(s):  
Robert Berkowitz ◽  
Heini Ilves ◽  
Wei Yu Lin ◽  
Karl Eckert ◽  
Andrea Coward ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Target Cell ◽  
Expression System ◽  
Cell Types ◽  
Human Cells ◽  
Bovine Immunodeficiency Virus ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Immunodeficiency Virus ◽  
Immortalized Cell

ABSTRACT Because lentiviruses are able to infect nondividing cells, these viruses might be utilized in gene therapy applications where the target cell does not divide. However, it has been suggested that the introduction of primate lentivirus sequences, particularly those of human immunodeficiency virus, into human cells may pose a health risk for the patient. To avoid this concern, we have constructed gene transfer systems based on a nonprimate lentivirus, bovine immunodeficiency virus. A panel of vectors and packaging constructs was generated and analyzed in a transient expression system for virion production and maturation, vector expression and encapsidation, and envelope protein pseudotyping. Virion preparations were also analyzed for transduction efficiency in a panel of human and nonhuman primary cells and immortalized cell lines. The virion preparations transduced most of the target cell types, with efficiencies up to 90% and with titers of unconcentrated virus up to 5 × 105infectious doses/ml. In addition, infection of nondividing human cells, including unstimulated hematopoietic stem cells and irradiated endothelial cells, was observed.

scholarly journals Cell Specific Dopamine Modulation of the Transient Potassium Current in the Pyloric Network by the Canonical D1 Receptor Signal Transduction Cascade

2010 ◽  
Vol 104 (2) ◽  
pp. 873-884 ◽  
Author(s):  
Hongmei Zhang ◽  
Edmund W. Rodgers ◽  
Wulf-Dieter C. Krenz ◽  
Merry C. Clark ◽  
Deborah J. Baro
Keyword(s):  
Potassium Current ◽  
Expression System ◽  
Expression Patterns ◽  
Motor Pattern ◽  
Cell Types ◽  
Receptor Expression ◽  
Intracellular Camp ◽  
Cell Type ◽  
Pyloric Network ◽  
Cell Type Specific

Dopamine (DA) modifies the motor pattern generated by the pyloric network in the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus , by directly acting on each of the circuit neurons. The 14 pyloric neurons fall into six cell types, and DA actions are cell type specific. The transient potassium current mediated by shal channels ( IA) is a common target of DA modulation in most cell types. DA shifts the voltage dependence of IA in opposing directions in pyloric dilator (PD) versus lateral pyloric (LP) neurons. The mechanism(s) underpinning cell-type specific DA modulation of IA is unknown. DA receptors (DARs) can be classified as type 1 (D1R) or type 2 (D2R). D1Rs and D2Rs are known to increase and decrease intracellular cAMP concentrations, respectively. We hypothesized that the opposing DA effects on PD and LP IA were due to differences in DAR expression patterns. In the present study, we found that LP expressed somatodendritic D1Rs that were concentrated near synapses but did not express D2Rs. Consistently, DA modulation of LP IA was mediated by a Gs-adenylyl cyclase-cAMP-protein kinase A pathway. Additionally, we defined antagonists for lobster D1Rs (flupenthixol) and D2Rs (metoclopramide) in a heterologous expression system and showed that DA modulation of LP IA was blocked by flupenthixol but not by metoclopramide. We previously showed that PD neurons express D2Rs, but not D1Rs, thus supporting the idea that cell specific effects of DA on IA are due to differences in receptor expression.

The fate of acetyl groups and sugar components during the digestion of grass cell walls in sheep

1977 ◽  
Vol 89 (2) ◽  
pp. 327-340 ◽  
Author(s):  
E. Jane Morris ◽  
J. S. D. Bacon
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Lignin Content ◽  
Sodium Ethoxide ◽  
Mesophyll Cell ◽  
Cell Types ◽  
Gas Liquid Chromatography ◽  
Cell Wall Preparation ◽  
The Relationship ◽  
Isolated Cell

SummaryThe digestibilities of grass cell wall constituents determined in a digestion trial were compared with those obtained by suspending various isolated cell wall preparations in nylon bags in the rumen of a sheep. Particular attention was paid to acetyl groups and to individual sugars, which were determined in both cases by gas liquid chromatography.For dried grass and hay in the digestion trial the cell wall constituents showed digestibilities decreasing in the following order: arabinose, galactose, glucose, xylose, acetyl, lignin.For a leaf cell wall preparation derived from all cell types except mesophyll, the nylon bag technique allowed the same order of digestibilities; rhamnose and uronic acids were also measured and found to be rapidly digested. Mesophyll cell walls placed in nylon bags were more readily digested than non-mesophyll. All the sugars, and also acetyl groups, were digested to the same extent.In a grass cell wall preparation isolated from sheep faeces, tested similarly, xylose and glucose were digested to the same extent, but acetyl groups were less digested.Removal of acetyl groups, using sodium ethoxide, which left the sugar composition and lignin content unchanged, increased the digestibility particularly of the cell walls from faeces.The results are discussed with reference to the relationship between cell wall composition and digestibility.

scholarly journals Brain-wide single neuron reconstruction reveals morphological diversity in molecularly defined striatal, thalamic, cortical and claustral neuron types

2020 ◽  
Author(s):  
Hanchuan Peng ◽  
Peng Xie ◽  
Lijuan Liu ◽  
Xiuli Kuang ◽  
Yimin Wang ◽  
...  
Keyword(s):  
Single Neuron ◽  
Morphological Diversity ◽  
Cell Types ◽  
Fluorescent Labeling ◽  
Computer Assisted ◽  
Large Set ◽  
Whole Brain ◽  
Wide Range ◽  
Neuronal Types ◽  
Organizational Rules

Abstract Ever since the seminal findings of Ramon y Cajal, dendritic and axonal morphology has been recognized as a defining feature of neuronal types. Yet our knowledge concerning the diversity of neuronal morphologies, in particular distal axonal projection patterns, is extremely limited. To systematically obtain single neuron full morphology on a brain-wide scale, we established a platform with five major components: sparse labeling, whole-brain imaging, reconstruction, registration, and classification. We achieved sparse, robust and consistent fluorescent labeling of a wide range of neuronal types by combining transgenic or viral Cre delivery with novel transgenic reporter lines. We acquired high-resolution whole-brain fluorescent images from a large set of sparsely labeled brains using fluorescence micro-optical sectioning tomography (fMOST). We developed a set of software tools for efficient large-volume image data processing, registration to the Allen Mouse Brain Common Coordinate Framework (CCF), and computer-assisted morphological reconstruction. We reconstructed and analyzed the complete morphologies of 1,708 neurons from the striatum, thalamus, cortex and claustrum. Finally, we classified these cells into multiple morphological and projection types and identified a set of region-specific organizational rules of long-range axonal projections at the single cell level. Specifically, different neuron types from different regions follow highly distinct rules in convergent or divergent projection, feedforward or feedback axon termination patterns, and between-cell homogeneity or heterogeneity. Major molecularly defined classes or types of neurons have correspondingly distinct morphological and projection patterns, however, we also identify further remarkably extensive morphological and projection diversity at more fine-grained levels within the major types that cannot presently be accounted for by preexisting transcriptomic subtypes. These insights reinforce the importance of full morphological characterization of brain cell types and suggest a plethora of ways different cell types and individual neurons may contribute to the function of their respective circuits.

scholarly journals Retroviral mediated transfer and expression of exogenous genes in primary lymphoid cells: assaying for a viral transactivator activity in normal and malignant cells

Blood ◽  
1990 ◽  
Vol 76 (6) ◽  
pp. 1201-1208
Author(s):  
RK Strair ◽  
M Towle ◽  
PW Heald ◽  
BR Smith
Keyword(s):  
Cell Types ◽  
Lymphoproliferative Disorders ◽  
Lymphoid Cells ◽  
Viral Gene ◽  
Specific Cell ◽  
Gene Products ◽  
Primary Cells ◽  
Viral Transactivator ◽  
Exogenous Genes ◽  
Beta Galactosidase

In this report we describe the use of recombinant retroviruses to characterize the activity of an exogenous promoter in primary cells obtained from patients with lymphoproliferative disorders. The infection of a variety of cultured and primary lymphoid cells with a recombinant retrovirus containing a histone promoter-driven beta- galactosidase gene is shown to result in the expression of beta- galactosidase in 50% to 100% of the cells. A similar infection with a recombinant retrovirus containing the beta-galactosidase gene with an adenovirus E2 promoter, results in beta-galactosidase activity in a limited number of cultured and primary cells. Since the adenovirus E2 promoter has been well characterized and is known to be regulated by transactivators encoded by many viruses, the activity of this promoter in specific cell types is discussed in reference to both the biology of the cell and the possible presence of as yet undetected viral gene products.

scholarly journals Brain-wide single neuron reconstruction reveals morphological diversity in molecularly defined striatal, thalamic, cortical and claustral neuron types

2019 ◽  
Author(s):  
Hanchuan Peng ◽  
Peng Xie ◽  
Lijuan Liu ◽  
Xiuli Kuang ◽  
Yimin Wang ◽  
...  
Keyword(s):  
Single Neuron ◽  
Morphological Diversity ◽  
Cell Types ◽  
Fluorescent Labeling ◽  
Computer Assisted ◽  
Large Set ◽  
Whole Brain ◽  
Wide Range ◽  
Neuronal Types ◽  
Organizational Rules

ABSTRACTEver since the seminal findings of Ramon y Cajal, dendritic and axonal morphology has been recognized as a defining feature of neuronal types. Yet our knowledge concerning the diversity of neuronal morphologies, in particular distal axonal projection patterns, is extremely limited. To systematically obtain single neuron full morphology on a brain-wide scale, we established a platform with five major components: sparse labeling, whole-brain imaging, reconstruction, registration, and classification. We achieved sparse, robust and consistent fluorescent labeling of a wide range of neuronal types by combining transgenic or viral Cre delivery with novel transgenic reporter lines. We acquired high-resolution whole-brain fluorescent images from a large set of sparsely labeled brains using fluorescence micro-optical sectioning tomography (fMOST). We developed a set of software tools for efficient large-volume image data processing, registration to the Allen Mouse Brain Common Coordinate Framework (CCF), and computer-assisted morphological reconstruction. We reconstructed and analyzed the complete morphologies of 1,708 neurons from the striatum, thalamus, cortex and claustrum. Finally, we classified these cells into multiple morphological and projection types and identified a set of region-specific organizational rules of long-range axonal projections at the single cell level. Specifically, different neuron types from different regions follow highly distinct rules in convergent or divergent projection, feedforward or feedback axon termination patterns, and between-cell homogeneity or heterogeneity. Major molecularly defined classes or types of neurons have correspondingly distinct morphological and projection patterns, however, we also identify further remarkably extensive morphological and projection diversity at more fine-grained levels within the major types that cannot presently be accounted for by preexisting transcriptomic subtypes. These insights reinforce the importance of full morphological characterization of brain cell types and suggest a plethora of ways different cell types and individual neurons may contribute to the function of their respective circuits.

scholarly journals Transcriptional terminators allow leak-free chromosomal integration of genetic constructs in cyanobacteria

2019 ◽  
Author(s):  
Ciarán L. Kelly ◽  
George M. Taylor ◽  
Aiste Satkute ◽  
Linda Dekker ◽  
John T. Heap
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Chromosomal Integration ◽  
Rna Structures ◽  
Independent Control ◽  
Poor Control ◽  
Control Of Gene Expression ◽  
Genomic Locations ◽  
Genetic Constructs ◽  
Control Of Expression

AbstractCyanobacteria are promising candidates for sustainable bioproduction of chemicals from sunlight and carbon dioxide. However, the genetics and metabolism of cyanobacteria are less well understood than model heterotrophic organisms, and the suite of well characterised cyanobacterial genetic tools and parts is less mature and complete. Transcriptional terminators use specific RNA structures to halt transcription and are routinely used in both natural and recombinant contexts to achieve independent control of gene expression and ‘insulate’ genes and operons from one another. Insulating gene expression can be particularly important when heterologous/synthetic genetic constructs are inserted at genomic locations where transcriptional read-through from chromosomal promoters occurs, resulting in poor control of expression of the introduced genes. To date, few terminators have been described and characterised in cyanobacteria. In this work, nineteen heterologous, synthetic or putative native Rho-independent (intrinsic) terminators were tested in the model freshwater cyanobacterium,Synechocystissp. PCC 6803, from which eleven strong terminators were identified. A subset of these strong terminators was then used to successfully insulate a chromosomally-integrated rhamnose-induciblerhaBADexpression system from hypothesised ‘read-through’ from a neighbouring chromosomal promoter, resulting in greatly improved inducible control. The addition of validated strong terminators to the cyanobacterial toolkit will allow improved independent control of introduced genes.

Sign in / Sign up

Export Citation Format

Share Document