Faculty Opinions recommendation of Single-copy insertion of transgenes in Caenorhabditis elegans.

2008 ◽  
Author(s):  
Michel Labouesse
Keyword(s):  
Caenorhabditis Elegans ◽  
Single Copy

scholarly journals Lack of pairing during meiosis triggers multigenerational transgene silencing in Caenorhabditis elegans

2015 ◽  
Vol 112 (20) ◽  
pp. E2667-E2676 ◽  
Author(s):  
Luciana E. Leopold ◽  
Bree N. Heestand ◽  
Soobin Seong ◽  
Ludmila Shtessel ◽  
Shawn Ahmed
Keyword(s):  
Caenorhabditis Elegans ◽  
Small Rna ◽  
Transgene Expression ◽  
Single Copy ◽  
Transgene Silencing ◽  
Meiotic Pairing ◽  
Homologous Chromosomes ◽  
Diverse Species ◽  
Secondary Sirna ◽  
Interfering Rna

Single-copy transgenes in Caenorhabditis elegans can be subjected to a potent, irreversible silencing process termed small RNA-induced epigenetic silencing (RNAe). RNAe is promoted by the Piwi Argonaute protein PRG-1 and associated Piwi-interacting RNAs (piRNAs), as well as by proteins that promote and respond to secondary small interfering RNA (siRNA) production. Here we define a related siRNA-mediated silencing process, termed “multigenerational RNAe,” which can occur for transgenes that are maintained in a hemizygous state for several generations. We found that transgenes that contain either GFP or mCherry epitope tags can be silenced via multigenerational RNAe, whereas a transgene that possesses GFP and a perfect piRNA target site can be rapidly and permanently silenced via RNAe. Although previous studies have shown that PRG-1 is typically dispensable for maintenance of RNAe, we found that both initiation and maintenance of multigenerational RNAe requires PRG-1 and the secondary siRNA biogenesis protein RDE-2. Although silencing via RNAe is irreversible, we found that transgene expression can be restored when hemizygous transgenes that were silenced via multigenerational RNAe become homozygous. Furthermore, multigenerational RNAe was accelerated when meiotic pairing of the chromosome possessing the transgene was abolished. We propose that persistent lack of pairing during meiosis elicits a reversible multigenerational silencing response, which can lead to permanent transgene silencing. Multigenerational RNAe may be broadly relevant to single-copy transgenes used in experimental biology and to shaping the epigenomic landscape of diverse species, where genomic polymorphisms between homologous chromosomes commonly result in unpaired DNA during meiosis.

scholarly journals Rapid self-selecting and clone-free integration of transgenes into engineered CRISPR safe harbor locations in Caenorhabditis elegans

2020 ◽  
Author(s):  
Zachary C. Stevenson ◽  
Megan J. Moerdyk-Schauwecker ◽  
Brennen Jamison ◽  
Patrick C. Phillips
Keyword(s):  
Caenorhabditis Elegans ◽  
Single Copy ◽  
False Positives ◽  
Model Organisms ◽  
Screening Methods ◽  
Safe Harbor ◽  
Guide Rna ◽  
C Elegans ◽  
Pcr Products ◽  
Transgenic Lines

AbstractPrecision genome editing for model organisms has revolutionized functional analysis and validation of a wide variety of molecular systems. To date, the capacity to insert transgenes into the model nematode Caenorhabditis elegans has focused on utilizing either transposable elements or CRISPR-based safe harbor strategies. These methods require laborious screening processes that often result in false positives from heritable extrachromosomal arrays or rely on co-CRISPR markers to identify likely edited individuals. As a result, verification of transgene insertion requires anti-array selection screening methods or extensive PCR genotyping respectively. These approaches also rely on cloning plasmids for the addition of transgenes. Here, we present a novel safe harbor CRISPR-based integration strategy that utilizes engineered insertion locations containing a synthetic guide RNA target and a split-selection system to eliminate false positives from array formation, thereby providing integration-specific selection. This approach allows the experimenter to confirm an integration event has taken place without molecular validation or anti-array screening methods, and is capable of producing integrated transgenic lines in as little as five days post-injection. To further increase the speed of generating transgenic lines, we also utilized the C. elegans native homology-based formation of extra-chromosomal arrays to assemble transgenes in-situ, removing the cloning step. We show that complete transgenes can be made and inserted into our split-selection safe harbor locations starting from PCR products, providing a clone-free and molecular-validation-free strategy for single-copy transgene integration. Overall, this combination of approaches provides an economical and rapid system for generating highly reproducible complex transgenics in C. elegans.

scholarly journals Single-copy Knock-In Loci for Defined Gene Expression in C. elegans

2018 ◽  
Author(s):  
Carlos G Silva-Garcia ◽  
Caroline Heintz ◽  
Sneha Dutta ◽  
Nicole M Clark ◽  
Anne Lanjuin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Cdna Sequence ◽  
Single Copy ◽  
Tissue Specific ◽  
C Elegans

We have generated a single-copy knock-in loci for defined gene expression (SKI LODGE) system to insert any cDNA by CRISPR/Cas9 at defined safe harbors in the Caenorhabditis elegans genome. Utilizing a single crRNA guide, which also acts as a Co-CRISPR enrichment marker, any cDNA sequence can be introduced as a single integrated copy, regulated by different tissue-specific promoters. The SKI LODGE system provides a fast, economical and effective approach for generating single-copy non-silenced transgenes in C. elegans.

scholarly journals Rapid Self-Selecting and Clone-Free Integration of Transgenes into Engineered CRISPR Safe Harbor Locations in Caenorhabditis elegans

2020 ◽  
Vol 10 (10) ◽  
pp. 3775-3782
Author(s):  
Zachary C. Stevenson ◽  
Megan J. Moerdyk-Schauwecker ◽  
Brennen Jamison ◽  
Patrick C. Phillips
Keyword(s):  
Caenorhabditis Elegans ◽  
Single Copy ◽  
Model Organisms ◽  
Screening Methods ◽  
Safe Harbor ◽  
Molecular Systems ◽  
Guide Rna ◽  
C Elegans ◽  
Pcr Products ◽  
Transgenic Lines

Precision genome editing for model organisms has revolutionized functional analysis and validation of a wide variety of molecular systems. To date, the capacity to insert single-copy transgenes into the model nematode Caenorhabditis elegans has focused on utilizing either transposable elements or CRISPR-based safe harbor strategies. These methods require plate-level screening processes to avoid selecting heritable extrachromosomal arrays or rely on co-CRISPR markers to identify knock-in events. As a result, verification of transgene insertion requires anti-array selection screening methods and PCR genotyping. These approaches also rely on cloning plasmids for the addition of transgenes. Here, we present a novel safe harbor CRISPR-based integration strategy that utilizes engineered insertion locations containing a synthetic guide RNA target and a split-selection system to eliminate false positives from array formation, thereby providing integration-specific selection. This approach allows the experimenter to confirm an integration event has taken place without molecular validation or anti-array screening methods and is capable of producing integrated transgenic lines in as little as five days post-injection. To further increase the speed of generating transgenic lines, we also utilized the C. elegans native microhomology-based recombination, to assemble transgenes in-situ, removing the cloning step. We show that complete transgenes can be made and inserted into our split-selection safe harbor locations starting from PCR products, providing a clone-free and molecular-validation-free strategy for single-copy transgene integration. Overall, this combination of approaches provides an economical and rapid system for generating highly reproducible complex transgenics in C. elegans.

scholarly journals elt-1, an embryonically expressed Caenorhabditis elegans gene homologous to the GATA transcription factor family.

1991 ◽  
Vol 11 (9) ◽  
pp. 4651-4659 ◽  
Author(s):  
J Spieth ◽  
Y H Shim ◽  
K Lea ◽  
R Conrad ◽  
T Blumenthal
Keyword(s):  
Transcription Factor ◽  
Caenorhabditis Elegans ◽  
Dna Binding ◽  
Single Copy ◽  
Amino Acid Sequences ◽  
Recognition Sequence ◽  
Sequence Element ◽  
C Elegans ◽  
Binding Domains ◽  
Degenerate Oligonucleotide

The short, asymmetrical DNA sequence to which the vertebrate GATA family of transcription factors binds is present in some Caenorhabditis elegans gene regulatory regions: it is required for activation of the vitellogenin genes and is also found just 5' of the TATA boxes of tra-2 and the msp genes. In vertebrates GATA-1 is specific to erythroid lineages, whereas GATA-2 and GATA-3 are present in multiple tissues. In an effort to identify the trans-acting factors that may recognize this sequence element in C. elegans, we used a degenerate oligonucleotide to clone a C. elegans homolog to this gene. We call this gene elt-1 (erythrocytelike transcription factor). It is single copy and specifies a 1.75-kb mRNA that is present predominantly, if not exclusively, in embryos. The region of elt-1 encoding two zinc fingers is remarkably similar to the DNA-binding domain of the vertebrate GATA-binding proteins. However, outside of the DNA-binding domains the amino acid sequences are quite divergent. Nevertheless, introns are located at identical or nearly identical positions in elt-1 and the mouse GATA-1 gene. In addition, elt-1 mRNA is trans-spliced to the 22-base untranslated leader, SL1. The DNA upstream of the elt-1 TATA box contains eight copies of the GATA recognition sequence within the first 300 bp, suggesting that elt-1 may be autogenously regulated. Our results suggest that the specialized role of GATA-1 in erythroid gene expression was derived after separation of the nematodes and the line that led to the vertebrates, since C. elegans lacks an erythroid lineage.

elt-1, an embryonically expressed Caenorhabditis elegans gene homologous to the GATA transcription factor family

1991 ◽  
Vol 11 (9) ◽  
pp. 4651-4659
Author(s):  
J Spieth ◽  
Y H Shim ◽  
K Lea ◽  
R Conrad ◽  
T Blumenthal
Keyword(s):  
Transcription Factor ◽  
Caenorhabditis Elegans ◽  
Dna Binding ◽  
Single Copy ◽  
Amino Acid Sequences ◽  
Recognition Sequence ◽  
Sequence Element ◽  
C Elegans ◽  
Binding Domains ◽  
Degenerate Oligonucleotide

The short, asymmetrical DNA sequence to which the vertebrate GATA family of transcription factors binds is present in some Caenorhabditis elegans gene regulatory regions: it is required for activation of the vitellogenin genes and is also found just 5' of the TATA boxes of tra-2 and the msp genes. In vertebrates GATA-1 is specific to erythroid lineages, whereas GATA-2 and GATA-3 are present in multiple tissues. In an effort to identify the trans-acting factors that may recognize this sequence element in C. elegans, we used a degenerate oligonucleotide to clone a C. elegans homolog to this gene. We call this gene elt-1 (erythrocytelike transcription factor). It is single copy and specifies a 1.75-kb mRNA that is present predominantly, if not exclusively, in embryos. The region of elt-1 encoding two zinc fingers is remarkably similar to the DNA-binding domain of the vertebrate GATA-binding proteins. However, outside of the DNA-binding domains the amino acid sequences are quite divergent. Nevertheless, introns are located at identical or nearly identical positions in elt-1 and the mouse GATA-1 gene. In addition, elt-1 mRNA is trans-spliced to the 22-base untranslated leader, SL1. The DNA upstream of the elt-1 TATA box contains eight copies of the GATA recognition sequence within the first 300 bp, suggesting that elt-1 may be autogenously regulated. Our results suggest that the specialized role of GATA-1 in erythroid gene expression was derived after separation of the nematodes and the line that led to the vertebrates, since C. elegans lacks an erythroid lineage.

scholarly journals Single-copy insertion of transgenes in Caenorhabditis elegans

2008 ◽  
Vol 40 (11) ◽  
pp. 1375-1383 ◽  
Author(s):  
Christian Frøkjær-Jensen ◽  
M Wayne Davis ◽  
Christopher E Hopkins ◽  
Blake J Newman ◽  
Jason M Thummel ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Single Copy

scholarly journals The Caenorhabditis elegans vitellogenin gene family includes a gene encoding a distantly related protein.

1985 ◽  
Vol 5 (10) ◽  
pp. 2495-2501 ◽  
Author(s):  
J Spieth ◽  
T Blumenthal
Keyword(s):  
Caenorhabditis Elegans ◽  
Gene Family ◽  
Single Copy ◽  
Egg Laying ◽  
Base Pairs ◽  
Gene Encoding ◽  
Yolk Proteins ◽  
C Elegans ◽  
Single Precursor ◽  
Vitellogenin Gene

While the nematode Caenorhabditis elegans is more primitive than most egg-laying organisms, it's vitellogenins, or yolk protein precursors, appear to be more complex. C. elegans oocytes accumulate two major classes of yolk proteins. The first consists of two polypeptides with an Mr of about 170,000 (yp170A and yp170B) encoded by a family of five closely related genes called vit-1 through vit-5. The second class consists of two smaller proteins with Mr values of 115,000 (yp115) and 88,000 (yp88) which are cut from a single precursor. Here we report the cloning and analysis of a single-copy gene (vit-6) that encodes this precursor. The lengths of the gene and its mRNA are about 5 X 10(3) base pairs. Like vit-1 through vit-5, vit-6 is expressed exclusively in adult hermaphrodites. Comparison of portions of the coding sequence indicates that vit-6 is distantly related to the vit-1 through vit-5 gene family. Thus, even though the two classes of yolk proteins are antigenically and physically distinct, they are encoded by a single highly diverged gene family.

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