Approaches for gene targeting and targeted gene expression in plants

Abstract Vitis vinifera, one of the most cultivated fruit crops, is susceptible to several diseases particularly caused by fungus and oomycete pathogens. In contrast, other Vitis species (American, Asian) display different degrees of tolerance/resistance to these pathogens, being widely used in breeding programs to introgress resistance traits in elite V. vinifera cultivars. Secondary metabolites are important players in plant defence responses. Therefore, the characterization of the metabolic profiles associated with disease resistance and susceptibility traits in grapevine is a promising approach to identify trait-related biomarkers. In this work, the leaf metabolic composition of eleven Vitis genotypes was analysed using an untargeted metabolomics approach. A total of 190 putative metabolites were found to discriminate resistant/partial resistant from susceptible genotypes. The biological relevance of discriminative compounds was assessed by pathway analysis. Several compounds were selected as promising biomarkers and the expression of genes coding for enzymes associated with their metabolic pathways was analysed. Reference genes for these grapevine genotypes were established for normalisation of candidate gene expression. The leucoanthocyanidin reductase 2 gene (LAR2) presented a significant increase of expression in susceptible genotypes, in accordance with catechin accumulation in this analysis group. Up to our knowledge this is the first time that metabolic constitutive biomarkers are proposed, opening new insights into plant selection on breeding programs.

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Epidermal Langerhans Cell-Targeted Gene Expression by a Dectin-2 Promoter

The Journal of Immunology ◽

10.4049/jimmunol.167.12.6893 ◽

2001 ◽

Vol 167 (12) ◽

pp. 6893-6900 ◽

Cited By ~ 39

Author(s):

Makoto Bonkobara ◽

Paul K. Zukas ◽

Sojin Shikano ◽

Shinichiro Nakamura ◽

Ponciano D. Cruz ◽

...

Keyword(s):

Gene Expression ◽

Langerhans Cell ◽

Targeted Gene Expression

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Targeted gene expression profiling of inverted papilloma and squamous cell carcinoma

International Forum of Allergy & Rhinology ◽

10.1002/alr.22882 ◽

2021 ◽

Author(s):

Charles C. L. Tong ◽

Mateusz Koptyra ◽

Pichai Raman ◽

Komal S. Rathi ◽

Namrata Choudhari ◽

...

Keyword(s):

Gene Expression ◽

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Gene Expression Profiling ◽

Squamous Cell ◽

Expression Profiling ◽

Inverted Papilloma ◽

Targeted Gene Expression

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Short DNA sequences inserted for gene targeting can accidentally interfere with off‐target gene expression

The FASEB Journal ◽

10.1096/fj.09-140749 ◽

2010 ◽

Vol 24 (6) ◽

pp. 1714-1724 ◽

Cited By ~ 56

Author(s):

Ingo D. Meier ◽

Christian Bernreuther ◽

Thomas Tilling ◽

John Neidhardt ◽

Yong Wee Wong ◽

...

Keyword(s):

Gene Expression ◽

Gene Targeting ◽

Dna Sequences ◽

Target Gene ◽

Target Gene Expression

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Pcsk6 mutant mice exhibit progressive loss of ovarian function, altered gene expression, and formation of ovarian pathology

Reproduction ◽

10.1530/rep-10-0451 ◽

2011 ◽

Vol 141 (3) ◽

pp. 343-355 ◽

Cited By ~ 5

Author(s):

Michelle L Mujoomdar ◽

Laura M Hogan ◽

Albert F Parlow ◽

Mark W Nachtigal

Keyword(s):

Gene Expression ◽

Ovarian Function ◽

Elevated Serum ◽

Cell Types ◽

Reproductive Capacity ◽

Wide Range ◽

Altered Gene ◽

Ovarian Pathology ◽

Targeted Gene Expression ◽

Benign Ovarian Tumors

Bioactivation of precursor proteins by members of the proprotein convertase (PC) family is essential for normal reproduction. ThePcsk6gene is a member of the PC family that is expressed in numerous ovarian cell types including granulosa cells and oocytes. We hypothesized that loss of PCSK6 would produce adverse effects in the mouse ovary. Mice incapable of expressing PCSK6 (Pcsk6tm1Rob) were obtained, and reproductive parameters (serum hormones, whelping interval, estrus cyclicity, and fertility) were compared toPcsk6+/+mice. WhilePcsk6tm1Robfemale mice are fertile, they manifest reduced reproductive capacity at an accelerated rate relative toPcsk6+/+mice. Reproductive senescence is typically reached by 9 months of age and is correlated with loss of estrus cyclicity, elevated serum FSH levels, and gross alterations in ovarian morphology. A wide range of ovarian morphologies were identified encompassing mild, such as an apparent reduction in follicle number, to moderate – ovarian atrophy with a complete absence of follicles – to severe, manifesting as normal ovarian structures replaced by benign ovarian tumors, including tubulostromal adenomas. Targeted gene expression profiling highlighted changes in RNA expression of molecules involved in processes such as steroidogenesis, gonadotropin signaling, transcriptional regulation, autocrine/paracrine signaling, cholesterol handling, and proprotein bioactivation. These results show that PCSK6 activity plays a role in maintaining normal cellular and tissue homeostasis in the ovary.

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Identification of Split-GAL4 Drivers and Enhancers That Allow Regional Cell Type Manipulations of the Drosophila melanogaster Intestine

Genetics ◽

10.1534/genetics.120.303625 ◽

2020 ◽

Vol 216 (4) ◽

pp. 891-903

Author(s):

Ishara S. Ariyapala ◽

Jessica M. Holsopple ◽

Ellen M. Popodi ◽

Dalton G. Hartwick ◽

Lily Kahsai ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Expression Patterns ◽

Cell Types ◽

Enteroendocrine Cells ◽

Epithelial Tissue ◽

Cell Type ◽

Cell Functions ◽

Distinct Subset ◽

Targeted Gene Expression

The Drosophila adult midgut is a model epithelial tissue composed of a few major cell types with distinct regional identities. One of the limitations to its analysis is the lack of tools to manipulate gene expression based on these regional identities. To overcome this obstacle, we applied the intersectional split-GAL4 system to the adult midgut and report 653 driver combinations that label cells by region and cell type. We first identified 424 split-GAL4 drivers with midgut expression from ∼7300 drivers screened, and then evaluated the expression patterns of each of these 424 when paired with three reference drivers that report activity specifically in progenitor cells, enteroendocrine cells, or enterocytes. We also evaluated a subset of the drivers expressed in progenitor cells for expression in enteroblasts using another reference driver. We show that driver combinations can define novel cell populations by identifying a driver that marks a distinct subset of enteroendocrine cells expressing genes usually associated with progenitor cells. The regional cell type patterns associated with the entire set of driver combinations are documented in a freely available website, providing information for the design of thousands of additional driver combinations to experimentally manipulate small subsets of intestinal cells. In addition, we show that intestinal enhancers identified with the split-GAL4 system can confer equivalent expression patterns on other transgenic reporters. Altogether, the resource reported here will enable more precisely targeted gene expression for studying intestinal processes, epithelial cell functions, and diseases affecting self-renewing tissues.

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Multiplexed, Targeted Gene Expression Profiling and Genetic Analysis on Electronic Microarrays

Clinical Chemistry ◽

10.1093/clinchem/48.11.1873 ◽

2002 ◽

Vol 48 (11) ◽

pp. 1873-1882 ◽

Cited By ~ 18

Author(s):

Elaine M Weidenhammer ◽

Brenda F Kahl ◽

Ling Wang ◽

Larry Wang ◽

Melanie Duhon ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Target Genes ◽

Mrna Targets ◽

Amplification Method ◽

Polymorphism Detection ◽

Targeted Gene Expression ◽

Electronic Microarray ◽

Electronic Field

Abstract Background: Electronic microarrays comprise independent microelectrode test sites that can be electronically biased positive or negative, or left neutral, to move and concentrate charged molecules such as DNA and RNA to one or more test sites. We developed a protocol for multiplexed gene expression profiling of mRNA targets that uses electronic field-facilitated hybridization on electronic microarrays. Methods: A multiplexed, T7 RNA polymerase-mediated amplification method was used for expression profiling of target mRNAs from total cellular RNA; targets were detected by hybridization to sequence-specific capture oligonucleotides on electronic microarrays. Activation of individual test sites on the electronic microarray was used to target hybridization to designated subsets of sites and allow comparisons of target concentrations in different samples. We used multiplexed amplification and electronic field-facilitated hybridization to analyze expression of a model set of 10 target genes in the U937 cell line during lipopolysaccharide-mediated differentiation. Performance of multiple genetic analyses (single-nucleotide polymorphism detection, gene expression profiling, and splicing isoform detection) on a single electronic microarray was demonstrated using the ApoE and ApoER2 genes as a model system. Results: Targets were detected after a 2-min hybridization reaction. With noncomplementary capture probes, no signal was detectable. Twofold changes in target concentration were detectable throughout the (∼64-fold) range of concentrations tested. Levels of 10 targets were analyzed side by side across seven time points. By confining electronic activation to subsets of test sites, polymorphism detection, expression profiling, and splicing isoform analysis were performed on a single electronic microarray. Conclusions: Microelectronic array technology provides specific target detection and quantification with advantages over currently available methodologies for targeted gene expression profiling and combinatorial genomics testing.

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