scholarly journals Improved In Situ Hybridization to HIV with RNA Probes Derived from PCR Products

1997 ◽  
Vol 45 (5) ◽  
pp. 721-727 ◽  
Author(s):  
Richard W. Cone ◽  
Erika Schlaepfer
Keyword(s):  
In Situ Hybridization ◽  
Rna Polymerase ◽  
Plasmid Dna ◽  
T7 Rna Polymerase ◽  
Rna Probes ◽  
Pcr Products ◽  
Rna Polymerase Promoter ◽  
Hiv 1 ◽  
Specific Mrna

These experiments tested the hypothesis that a pool of PCR-derived RNA probes with defined length and even representation of the target sequences could produce more specific and intense in situ hybridization signals than randomly size-reduced, plasmid-derived RNA probes. In situ hybridization was performed with sense and anti-sense HIV-1 RNA probes that were derived from PCR products tailed with the T7 RNA polymerase promoter or from plasmid DNA. In situ hybridization using a pool of seven anti-sense or sense PCR-derived RNA probes (1805 nucleotides of HIV sequence, 257 nucleotides average probe length) was compared with hybridization using anti-sense or sense RNA probes made from a plasmid representing the HIV-1 env gene (3151 nucleotides of HIV-1 target). The pooled PCR-derived probes resulted in stronger in situ hybridization signals and less background than those produced with plasmid-derived RNA probes. This method for creating PCR-derived RNA probes improves the feasibility of synthesizing multiple, discrete RNA probes for studies of specific mRNA expression because it does not require the subcloning steps used to construct plasmids. PCR-derived RNA probes may provide a viable alternative to the use of plasmid-derived RNA probes for in situ hybridization.

scholarly journals PCR-derived ssDNA Probes for Fluorescent In Situ Hybridization to HIV-1 RNA

2000 ◽  
Vol 48 (2) ◽  
pp. 285-293 ◽  
Author(s):  
Marlyse C. Knuchel ◽  
Brigit Graf ◽  
Erika Schlaepfer ◽  
Herbert Kuster ◽  
Marek Fischer ◽  
...  
Keyword(s):  
T Cells ◽  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Uracil Dna Glycosylase ◽  
Rna Probes ◽  
Immunodeficiency Virus ◽  
Polymerase Chain ◽  
Rna Probe ◽  
Hiv 1

We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 μg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH.

scholarly journals A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei

2020 ◽  
Author(s):  
Sebastian Shaw ◽  
Sebastian Knüsel ◽  
Sarah Hoenner ◽  
Isabel Roditi
Keyword(s):  
Rna Polymerase ◽  
Cell Line ◽  
Genome Editing ◽  
Trypanosoma Brucei ◽  
Transient Expression ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
A Genome ◽  
Pcr Products

Abstract Objective Generation of knockouts and in situ tagging of genes in Trypanosoma brucei has been greatly facilitated by using CRISPR/Cas9 as a genome editing tool. To date, this has entailed using a limited number of cell lines that are stably transformed to express Cas9 and T7 RNA polymerase (T7RNAP). It would be desirable, however, to be able to use CRISPR/Cas9 for any trypanosome cell line. Results We describe a sequential transfection expression system that enables transient expression of the two proteins, followed by delivery of PCR products for gRNAs and repair templates. This procedure can be used for genome editing without the need for stable integration of the Cas9 and T7RNAP genes.

scholarly journals A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei

2020 ◽  
Author(s):  
Sebastian Shaw ◽  
Sebastian Knüsel ◽  
Sarah Hoenner ◽  
Isabel Roditi
Keyword(s):  
Rna Polymerase ◽  
Genome Editing ◽  
Trypanosoma Brucei ◽  
Transient Expression ◽  
Cell Lines ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
A Genome ◽  
Pcr Products

Abstract ObjectiveGeneration of knockouts and in situ tagging of genes in Trypanosoma brucei has been greatly facilitated by using CRISPR/Cas9 as a genome editing tool. To date, this has entailed using a limited number of cell lines that are stably transformed to express Cas9 and T7 RNA polymerase (T7RNAP). It would be desirable, however, to be able to use CRISPR/Cas9 for any cell line.ResultsWe describe a sequential transfection expression system that enables transient expression of the two proteins, followed by delivery of PCR products for gRNAs and repair templates. This procedure can be used for genome editing without the need for stable integration of the Cas9 and T7RNAP genes.

Tests of a model of specific contacts in T7 RNA polymerase-promoter interactions

Biochemistry ◽  
1995 ◽  
Vol 34 (2) ◽  
pp. 666-672 ◽  
Author(s):  
Charlie Schick ◽  
Craig T. Martin
Keyword(s):  
Rna Polymerase ◽  
T7 Rna Polymerase ◽  
Rna Polymerase Promoter

In situ hybridization reveals differential spatial distribution of mRNAs for type I and type II collagen in the chick limb bud

Development ◽  
1988 ◽  
Vol 103 (1) ◽  
pp. 111-118 ◽  
Author(s):  
C.J. Devlin ◽  
P.M. Brickell ◽  
E.R. Taylor ◽  
A. Hornbruch ◽  
R.K. Craig ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Limb Development ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Limb Bud ◽  
Type I ◽  
Type Ii ◽  
Mrna Accumulation ◽  
Rna Probes

During limb development, type I collagen disappears from the region where cartilage develops and synthesis of type II collagen, which is characteristic of cartilage, begins. In situ hybridization using antisense RNA probes was used to investigate the spatial localization of type I and type II collagen mRNAs. The distribution of the mRNA for type II collagen corresponded well with the pattern of type II collagen synthesis, suggesting control at the level of transcription and mRNA accumulation. In contrast, the pattern of mRNA for type I collagen remained more or less uniform and did not correspond with the synthesis of the protein, suggesting control primarily at the level of translation or of RNA processing.

scholarly journals Reassessment of the Roles of Integrase and the Central DNA Flap in Human Immunodeficiency Virus Type 1 Nuclear Import

2002 ◽  
Vol 76 (23) ◽  
pp. 12087-12096 ◽  
Author(s):  
Jeffrey D. Dvorin ◽  
Peter Bell ◽  
Gerd G. Maul ◽  
Masahiro Yamashita ◽  
Michael Emerman ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
In Situ Hybridization ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Immunodeficiency Virus ◽  
Central Polypurine Tract ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) can infect nondividing cells productively because the nuclear import of viral nucleic acids occurs in the absence of cell division. A number of viral factors that are present in HIV-1 preintegration complexes (PICs) have been assigned functions in nuclear import, including an essential valine at position 165 in integrase (IN-V165) and the central polypurine tract (cPPT). In this article, we report a comparison of the replication and infection characteristics of viruses with disruptions in the cPPT and IN-V165. We found that viruses with cPPT mutations still replicated productively in both dividing and nondividing cells, while viruses with a mutation at IN-V165 did not. Direct observation of the subcellular localization of HIV-1 cDNAs by fluorescence in situ hybridization revealed that cDNAs synthesized by both mutant viruses were readily detected in the nucleus. Thus, neither the cPPT nor the valine residue at position 165 of integrase is essential for the nuclear import of HIV-1 PICs.

Analysis of HIV-1 expression in vivo with in situ hybridization and the polymerase chain reaction

1992 ◽  
Vol 6 (3) ◽  
pp. 215-221 ◽  
Author(s):  
B. Delord ◽  
M. Ottmann ◽  
M.-H. Schrive ◽  
J.-M. Ragnaud ◽  
J.-M. Seigneurin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
In Situ Hybridization ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Hiv 1

scholarly journals Localization of cells producing erythropoietin in murine liver by in situ hybridization [see comments]

Blood ◽  
1991 ◽  
Vol 77 (11) ◽  
pp. 2497-2503 ◽  
Author(s):  
ST Koury ◽  
MC Bondurant ◽  
MJ Koury ◽  
GL Semenza
Keyword(s):  
In Situ Hybridization ◽  
Transgenic Mice ◽  
Antisense Rna ◽  
Interstitial Cells ◽  
Cell Type ◽  
Isolated Cells ◽  
Rna Probes ◽  
Sense Strand ◽  
Murine Liver

Abstract In situ hybridization using antisense RNA probes was used to localize cells that produce erythropoietin (EPO) in the livers of anemic transgenic mice expressing the human EPO gene and in livers of anemic nontransgenic mice. In transgenic mice bled from a hematocrit of 55% to one of 10%, hepatocytes surrounding central veins synthesized large amounts of human EPO mRNA. EPO-producing cells were very rare in the area of portal triads. In transgenic mice bled to a hematocrit of 20%, a similar number and distribution of cells contained human EPO mRNA as was found with a 10% hematocrit, but the cells were less heavily labeled, indicating increased EPO production per cell at 10% hematocrit as compared with 20% hematocrit. No human EPO mRNA was detected in the kidneys of anemic transgenic mice, although endogenous murine EPO mRNA was strongly expressed in cortical interstitial cells. In sections of livers from nontransgenic mice bled from a hematocrit of 45% to one of 10%, only isolated cells produced EPO. When the types of cells could clearly be identified, approximately 80% of these cells were hepatocytes, while 20% had a nonepithelial morphology and were located in or adjacent to the sinusoidal spaces. When the sense strand was used as the RNA probe for in situ hybridization, no labeled cells were seen in normal or anemic livers. These results demonstrate that hepatocytes are responsible for production of EPO in both transgenic and nontransgenic mice and that a second cell type that is similar in morphology to EPO-producing interstitial cells in the kidney also produces EPO in the livers of nontransgenic mice.

Sign in / Sign up

Export Citation Format

Share Document