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Internal Ribosome Entry Sites
Internal Ribosome Entry Sites
Internal Ribosome Entry Sites (IRES) are specific RNA sequence elements that allow for cap-indepedent translation initiation by recruiting ribosomes to the mRNA.
IRES are commonly found in the 5' untranslated regions (UTRs) of viral mRNAs, but can also occur in cellular mRNAs.
They enable the translation of mRNAs in a cap-independent manner, which is beneficial for viral infection and cellular stress responses.
Optimizing IRES sequence and structure is crucial for enhancing gene expression in biotechnology and reasearch applications.
PubCompare.ai provides an AI-driven platform to discover and compare the most effective IRES protocols from scientific literature, preprints, and patents, streamlining IRES research and protocol optimizaton.
IRES are commonly found in the 5' untranslated regions (UTRs) of viral mRNAs, but can also occur in cellular mRNAs.
They enable the translation of mRNAs in a cap-independent manner, which is beneficial for viral infection and cellular stress responses.
Optimizing IRES sequence and structure is crucial for enhancing gene expression in biotechnology and reasearch applications.
PubCompare.ai provides an AI-driven platform to discover and compare the most effective IRES protocols from scientific literature, preprints, and patents, streamlining IRES research and protocol optimizaton.
Most cited protocols related to «Internal Ribosome Entry Sites»
Alleles
Biological Assay
Brain
Genetic Background
Internal Ribosome Entry Sites
Intersectional Framework
Mice, Laboratory
Microscopy
Microscopy, Confocal
Recombination, Genetic
Internal Ribosome Entry Sites
Mice, Laboratory
Constructs used to produce AAV included pGP-AAV-syn-GCaMP-WPRE and the Cre recombinase-activated construct pGP-AAV-syn-flex-GCaMP-WPRE. Virus was injected slowly (30 nL in 5 minutes) at a depth of 250 μm into the primary visual cortex (two sites, 2.5 and 2.9 mm lateral from the lambda suture). For population imaging and electrophysiology (Fig 2 -3 ), AAV2/1-syn-GCaMP-WPRE virus (titer: ∼1011 (link) -1012 (link) genomes/mL) was injected into the visual cortex of C57BL/6J mice (1.5-2 months old)6 (link). For dendritic imaging (Fig 4 , 5 and 6a-f ), sparse labeling was achieved by injecting a mixture of diluted AAV2/1-syn-Cre particles (titer: ∼1012 (link) genomes/mL, diluted 8000-20,000 fold in PBS) and high titer, Cre-dependent GCaMP6s virus (∼8×1011 (link) genomes/mL). This produces strong GCaMP6 expression in a small subset of neurons (∼3-5 cells in a 250 μm × 250 μm × 250 μm volume), defined by Cre expression56 (link). Both pyramidal (Fig. 4 -5 ) and GABAergic (Fig. 6 ) neurons were labeled using this approach, but they could be distinguished based on the presence or absence of dendritic spines. Post hoc immunolabeling further identified the imaged cells. For specific labeling of parvalbumin interneurons (Fig. 6g and Supplementary Fig. 12 ), Cre-dependent GCaMP6s AAV was injected into the visual cortex of PV-IRES-Cre mice57 (link). Individual somata (Supplementary Fig. 12 ) and dendritic segments could be recognized (Fig. 6 g, h , total length of imaged dendrite: 2.86 mm), but the high labeling density made it difficult to track individual dendrites over long distances.
Cells
Cre recombinase
Dendrites
Dendritic Spines
Genome
Internal Ribosome Entry Sites
Interneurons
Mice, Inbred C57BL
Neurons
Parvalbumins
Striate Cortex
Sutures
TCL1B protein, human
Virus
Visual Cortex
Isl1 genomic DNA had been previously isolated from a mouse 129/Sv genomic library (Stratagene) as described by Pfaff et al. [25 (link)]. A PacI site had been introduced into an EcoRI site in the exon encoding the second LIM domain of Isl1. A cassette coating IRES Cre SV40 pA and pgk-neomycin was cloned into this PacI site to create a targeting construct with flanking 5' and 3' genomic DNA arms of 5 kb and 2 kb, respectively. ES cells were targeted and screened as described in Pfaff et al.[25 (link)].
Arm, Upper
Deoxyribonuclease EcoRI
Embryonic Stem Cells
Exons
Genome
Genomic Library
Internal Ribosome Entry Sites
Mice, 129 Strain
Neomycin
Simian virus 40
Er81EWS-Pea3 mice were generated following a strategy similar to that described for the generation of Er81NLZ mice [14 (link)]. In brief, a targeting vector with a cDNA coding for EWS-Pea3 was inserted in frame with the endogenous start ATG into exon 2 of the Er81 genomic locus and used for homologous recombination in W95 ES cells. EWS-Pea3 represents a fusion gene between the amino terminal of EWS and the ETS domain of Pea3 [20 (link)]. The primer pair used to specifically detect the Er81EWS-Pea3 allele was 5′-
CAGCCACTGCACCTACAAGAC-3′ and 5′-
CTTCCTGCTTGATGTCTCCTTC-3′. For the generation of TaumGFP and TauEWS-Pea3 mice, lox-STOP-lox-mGFP-IRES-NLS-LacZ-pA and lox-STOP-lox-EWS-Pea3-IRES-NLS-LacZ-pA targeting cassettes were integrated into exon 2 of the Tau genomic locus (the endogenous start ATG was removed in the targeting vectors; details available upon request). mGFP was provided by P. Caroni [25 (link)]. ES cell recombinants were screened by Southern blot analysis using the probe in the 5′ region as described previously [41 (link)]. Frequency of recombination in 129/Ola ES cells was approximately 1/3 for both Tau constructs. For the generation of PVCre mice, mouse genomic clones were obtained by screening a 129SV/J genomic library (Incyte, Wilmington, Delaware, United States). For details on the genomic structure of the mouse PV locus see [42 (link)]. An IRES-Cre-pA targeting cassette [33 (link)] was integrated into the 3′ UTR of exon 5, and ES cell recombinants were screened with a 5′ probe (oligos, 5′-
GAGATGACCCAGCCAGGATGCCTC-3′ and 5′-
CTGACCACTCTCGCTCCGGTGTCC-3′; genomic DNA, HindIII digest). The frequency of recombination in 129/Ola ES cells was approximately 1/20. Recombinant clones were aggregated with morula stage embryos to generate chimeric founder mice that transmitted the mutant alleles. In all experiments performed in this study, animals were of mixed genetic background (129/Ola and C57Bl6). Thy1spGFP transgenic mice were generated in analogy to De Paola et al. [25 (link)], and for these experiments a strain of mice with early embryonic expression was selected. Isl1Cre and Hb9Cre mouse strains have been described [33 (link),43 (link)] and Bax+/− animals were from Jackson Laboratory (Bar Harbor, Maine, United States) [27 (link)]. Timed pregnancies were set up to generate embryos of different developmental stages with all genotypes described throughout the study.
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2',5'-oligoadenylate
Alleles
Animals
Chimera
Clone Cells
Cloning Vectors
DNA, Complementary
Embryo
Embryonic Development
Embryonic Stem Cells
ETS Motif
Exons
Gene Fusion
Genetic Background
Genome
Genomic Library
Genotype
Homologous Recombination
Internal Ribosome Entry Sites
LacZ Genes
Mice, Laboratory
Mice, Transgenic
Morula
Oligonucleotide Primers
Pregnancy
Reading Frames
Recombination, Genetic
Southern Blotting
Strains
tau-1 monoclonal antibody
transcription factor PEA3
Most recents protocols related to «Internal Ribosome Entry Sites»
Example 2
The full-length murine NKG2D cDNA was purchased from Open Biosystems (Huntsville, AL). Murine CD3ζ chain, Dap10 and Dap12 cDNAs were cloned by RT-PCR using RNAs from ConA- or IL-2 (1000 U/mL)-activated spleen cells as templates. Mouse NKG2D ligands Rae-1p and H60 were cloned from YAC-1 cells by RT-PCR. All PCR reactions were performed using high-fidelity enzyme Pfu or PFUULTRA™ (STRATAGENE@, La Jolla, CA). The oligonucleotides employed in these PCR reactions are listed in Table 9.
Restriction sites inserted for cloning purposes are underlined.
Chimeric NKG2D was created by fusing the murine CD3 chain cytoplasmic region coding sequence (CD3′-CYP) to the full-length gene of murine NKG2D. Briefly, the SalI-EcoRI fragment of CD3′-CYP (with the initiation codon ATG at the 5′ end, primer numbers 9 and 10) and the EcoRI-XhoI fragment of NKG2D (without ATG, primer numbers 2 and 3) were ligated into the SalI/XhoI-digested pFB-neo retroviral vector (STRATAGENE®, La Jolla, CA). Similarly, chimeric Dap10 was generated by fusing the SalI-EcoRI fragment of full-length Dap10 (primer numbers 4 and 6) to the EcoRI-XhoI fragment of CD3ζ-CYP (primer numbers 11 and 12). Wild-type NKG2D (primer numbers 2 and 3), Dap10 (primer numbers 4 and 5) and Dap12 (primer numbers 7 and 8) fragments were inserted between the EcoRI and XhoI sites in pFB-neo. In some cases, a modified vector pFB-IRES-GFP was used to allow co-expression of green fluorescent protein (GFP) with genes of interest. pFB-IRES-GFP was constructed by replacing the 3.9 kb AvrUScaI fragment of pFB-neo with the 3.6kb AvrII/ScaI fragment of a plasmid GFP-RV(Ouyang, et al. (1998) Immunity 9:745-755). Rae-1β (primer numbers 13 and 14) and H60 (primer numbers 15 and 16) cDNAs were cloned into pFB-neo. Constructs containing human NKD2D and human CD3ζ or murine Fc were prepared in the same manner using the appropriate cDNAs as templates.
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Cells
Chimera
Cloning Vectors
Codon, Initiator
Concanavalin A
Cytoplasm
Deoxyribonuclease EcoRI
DNA, Complementary
Enzymes
Genes
Green Fluorescent Proteins
Homo sapiens
Internal Ribosome Entry Sites
Ligands
Mus
Oligonucleotide Primers
Oligonucleotides
Open Reading Frames
Plasmids
Response, Immune
Retroviridae
Reverse Transcriptase Polymerase Chain Reaction
RNA
Spleen
Example 2
Another example of a suitable vector is a retroviral vector. Retroviruses are RNA viruses that contain an RNA genome. The gag, pol, and env genes are flanked by long terminal repeat (LTR) sequences (or their corresponding proteins). The 5′ and 3′ LTR sequences promote transcription and polyadenylation of mRNAs.
The retroviral vector may provide a regulable transactivating element, an internal ribosome reentry site (IRES), a selection marker, and a target heterologous gene operated by a regulable promoter.
Alternatively, multiple sequences may be expressed under the control of multiple promoters. Finally, the retroviral vector may contain cis-acting sequences necessary for reverse transcription and integration. Upon infection, the RNA is reverse transcribed to DNA that integrates efficiently into the host genome. The recombinant retrovirus of this invention is genetically modified in such a way that some of the retroviral, infectious genes of the native virus have been removed and in certain instances replaced instead with a target nucleic acid sequence for genetic modification of the cell. The sequences may be exogenous DNA or RNA, in its natural or altered form.
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Base Sequence
Cells
Cloning Vectors
Electroporation
Gene Editing
Genes
Genes, env
Genes, Viral
Genome
Infection
Internal Ribosome Entry Sites
Long Terminal Repeat
Polyadenylation
Proteins
Retroviridae
Retroviridae Infections
Reverse Transcription
Ribosomes
RNA, Messenger
RNA Viruses
Transcription, Genetic
PERK.K622A.9E10.pCDNA myc tagged (21815; Addgene plasmid), PERK.WT.9E10.pCDNA myc tagged (21814; Addgene plasmid), eGFP-E-Syt1 (66830; Addgene plasmid) p-eGFP-C3 empty vector plasmid (2489; Addgene plasmid) and pmCherry-C1 empty vector plasmid (3552; Addgene plasmid) were obtained from Addgene. mCherry-E-Syt1, eGFP-E-Syt1-ΔDE, eGFP-E-Syt1-ΔSMP, and mCherry-E-Syt1-ΔCDE were a kind gift from Pietro De Camilli lab (Saheki et al., 2016 (link)). KDEL-HRP-myc was obtained from Francesca Giordano (Galmes et al., 2016 (link)). eGFP-E-Syt1-ΔCDE plasmid was generated from mCherry-E-Syt1-ΔCDE, E-Syt1-ΔCDE was excised using SalI and SacII restriction enzymes and cloned into p-eGFP-C3 empty vector (5′-E-Syt1-ΔCDE -SalI-FW cloning primer: 5′-TATATAGTCGACATGGAGCGATCTCCAGGAGA-3′; 5′ E-Syt1- ΔCDE -SacII-Rev cloning primer: 5′-TATATACCGCGGTCGAGGTGGGGCATCCACA-3′). mtAEQ WT was a kind gift from Paolo Pinton (University of Ferrara, Ferrara, Italy; Robert et al., 2000 (link)). pcDNA 3.1 mCherry-STIM1 was a gift from Peter Vangheluwe. Human PERK-myc WT cDNA (µHsPERK-myc-pUC57 SC1010) was generated by GenScript and cloned into a lentiviral transfer plasmid containing ires-hygromycin (pCHMWs-HsPERK-myc-Ires-Hygro SC1017) to select for transduced cells with hygromycin. The K622A mutant was introduced using a gBlock gene fragment (IDT) by replacing a part from the human PERK-myc WT by a fragment that contains the mutation. This construct also allows selection of transduced cells with hygromycin.
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Cells
Cloning Vectors
DNA, Complementary
DNA Restriction Enzymes
Genes
Homo sapiens
hygromycin A
Internal Ribosome Entry Sites
Mutation
Oligonucleotide Primers
Plasmids
STIM1 protein, human
SYT1 protein, human
The open reading frames of INPPL1 (GenScript: OHu19348), MAP4K5 (GenScript: OHu02673), and various mutants of these genes were cloned into pLV-EF1a-IRES-Neo (85139; Addgene) using a Gibson Assembly kit (E5510S; NEB). All sgRNAs were cloned into lentiCRISPRv2 (52961; Addgene) by following a published protocol (Sanjana et al., 2014 (link); Shalem et al., 2014 (link)). Lentiviruses were produced by co-transfecting HEK293T cells with viral construct and packaging vector DNAs (Hart et al., 2017 (link); Mair et al., 2019 (link)). Cells were spin-infected with concentrated viruses and infected cells were recovered by selection with the appropriate antibiotics.
Antibiotics
Cells
Cloning Vectors
DNA
Genes
Internal Ribosome Entry Sites
Lentivirus
Open Reading Frames
Virus
Where indicated, BMDMs were transduced with either a control vector, wild-type human MMP9, a catalytically inactive MMP9 E402/A mutant (Orgaz et al., 2014 (link)), wild-type human MMP14, a catalytically inactive MMP14 E240/A mutant expression vector (Shimizu-Hirota et al., 2012 (link)), or a constitutively active human RhoA L63 expression vector (RTV-204; Cell Biolabs). The MMP9 and MMP14 constructs were cloned into the pLentilox-IRES-GFP lentiviral vector, while the RhoA constructs were inserted into pBABEhygro retroviral vector. Expression of the recombinant proteins was confirmed by Western blot.
Cells
Cloning Vectors
Homo sapiens
Internal Ribosome Entry Sites
MMP9 protein, human
MMP14 protein, human
Recombinant Proteins
Retroviridae
RHOA protein, human
Western Blot
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Puromycin is a laboratory product manufactured by Merck Group. It functions as an antibiotic that inhibits protein synthesis in eukaryotic cells.
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PsPAX2 is a packaging plasmid used for the production of lentiviral particles. It contains the necessary genes for lentiviral packaging, but does not contain the viral genome. PsPAX2 is commonly used in lentiviral production workflows.
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C57BL/6J is a mouse strain commonly used in biomedical research. It is a common inbred mouse strain that has been extensively characterized.
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Penicillin/streptomycin is a commonly used antibiotic solution for cell culture applications. It contains a combination of penicillin and streptomycin, which are broad-spectrum antibiotics that inhibit the growth of both Gram-positive and Gram-negative bacteria.
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DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium formulated to support the growth and maintenance of a variety of cell types, including mammalian cells. It provides essential nutrients, amino acids, vitamins, and other components necessary for cell proliferation and survival in an in vitro environment.
More about "Internal Ribosome Entry Sites"
IRES, internal ribosome entry site, cap-independent translation, viral mRNA, cellular mRNA, biotechnology, research, protocol optimization, PubCompare.ai, Lipofectamine, Polybrene, Puromycin, FBS, PsPAX2, PMD2.G, C57BL/6J, Penicillin/streptomycin, DMEM