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A. A working model for DNA breakage resulting from L1 retrotranspostion. L1 retrotransposition occurs via target-primed reverse transcription. L1 ORF2p can nick genomic DNA at an EN target sequence (3’-AA/TTTT-5’) to liberate a polyT sequence that complementary bind the L1 RNA polyA sequence to form a primer-template structure. L1 ORF2p can then polymerize from the free 3’OH and use the L1 RNA as a template to generate de novo L1 cDNA that can be processed into a double-stranded insertion in the genome. Alternatively, DNA breakage can occur during the resolution of the L1 cDNA intermediate into an insertion generating recombinogenic DNA break ends. B. A schematic of the classic <t>L1-GFP</t> reporter assay. A pCEP4-Puro plasmid constitutively expresses a native human L1 sequence (L1RP) expressing ORF1p (RNA binding protein) and ORF2p (endonuclease and reverse transcriptase) containing a GFP reporter in the 3’ UTR that marks de novo L1 insertions. The GFP cassette is in the anti-sense direction relative to L1 sequence and is interrupted by an intron in the sense direction. The continuous GFP sequence is generated when the L1 transgene is transcribed and spliced but cannot be expressed and translated until the cassette is retrotransposed. Accordingly, GFP expression marks cells with de novo L1 insertions. C. Shown is the retrotransposition frequency of WT, reverse transcriptase mutant (RTmut, D702Y) RTmut or endonuclease mutant (ENmut, H230A) L1 measured by the L1-GFP reporter assay in U2OS cells. n = 6. P-value <0.0001, each compared to RTmut using one-way ANOVA with Dunnett’s test. D. Shown is a schematic of a new GFP reporter assay modeling the recombination between a L1-cDNA intermediate and a chromosomal break to generate a chromosomal rearrangement. Left: A pCEP4-Puro plasmid contains a modified version of the classic L1-GFP reporter construct, where the 3’ UTR contains an anti-sense 5’ GFP fragment (5’GFp) that is interrupted by a sense intron (L1-5’ GFp plasmid). Transcription, splicing and retrotransposition of the L1 RNA results in a canonical insertion containing an incomplete GFP cassette and does not induce GFP expression. Nonetheless, retrotransposition of the L1 RNA generates L1 insertion intermediates containing an 5’GFP fragment with the potential to be resolved in non-canonical events. Right: A 3’ GFP fragment containing 150 bp homology to the 5’ GFP fragment from L1-5’ GFp plasmid (3’ gFP 150bp Hom) was introduced chromosomally in U2OS cells using <t>the</t> <t>FRT/</t> Flp recombinase system (150bpHom-3’ gFP U2OS cells). A chromosomal break can be induced by the CRISRP/Cas9 system at the edge of the 150bp homology of the 3’gFP 150bp Hom sequence (Cas9/sg150bp). Hence, L1 cDNA insertion intermediates containing a 5’ GFP fragment can recombine with a broken chromosome containing a 3’ GFP fragment forming a chromosomal rearrangement and restoring GFP expression. E. Shown are representative flow cytometry plots of 150bpHom-3’ gFP U2OS cells transfected with WT or RTmut L1-5’ GFp reporter plasmid, and Cas9/ sgCTRL or Cas9/ sg150bp expressing plasmid. GFP expression is only induced with transfection of the WT L1-5’ GFp reporter plasmid and the Cas9/150bp expressing plasmid. F. Shown is the GFP frequency induced in U2OS FRT 3’ gFP (150bp Hom) cells transfected with WT, RTmut or ENmut L1-5’ GFp reporter plasmid, and Cas9/sgCTRL or Cas9/sg150bp expressing plasmid. n = 6. P-value calculated using unpaired two-tailed Student’s t tests with the Holm-Sidak correction for multiple comparisons. G. Shown is the GFP frequency induced in HEK293 150bpHom-3’ gFP cells transfected with WT, RTmut or ENmut L1-5’ GFp reporter plasmid, and Cas9/sgCTRL or Cas9/sg150bp expressing plasmid. n = 3. P-value calculated using unpaired two-tailed Student’s t tests with the Holm-Sidak correction for multiple comparisons.
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A. A working model for DNA breakage resulting from L1 retrotranspostion. L1 retrotransposition occurs via target-primed reverse transcription. L1 ORF2p can nick genomic DNA at an EN target sequence (3’-AA/TTTT-5’) to liberate a polyT sequence that complementary bind the L1 RNA polyA sequence to form a primer-template structure. L1 ORF2p can then polymerize from the free 3’OH and use the L1 RNA as a template to generate de novo L1 cDNA that can be processed into a double-stranded insertion in the genome. Alternatively, DNA breakage can occur during the resolution of the L1 cDNA intermediate into an insertion generating recombinogenic DNA break ends. B. A schematic of the classic L1-GFP reporter assay. A pCEP4-Puro plasmid constitutively expresses a native human L1 sequence (L1RP) expressing ORF1p (RNA binding protein) and ORF2p (endonuclease and reverse transcriptase) containing a GFP reporter in the 3’ UTR that marks de novo L1 insertions. The GFP cassette is in the anti-sense direction relative to L1 sequence and is interrupted by an intron in the sense direction. The continuous GFP sequence is generated when the L1 transgene is transcribed and spliced but cannot be expressed and translated until the cassette is retrotransposed. Accordingly, GFP expression marks cells with de novo L1 insertions. C. Shown is the retrotransposition frequency of WT, reverse transcriptase mutant (RTmut, D702Y) RTmut or endonuclease mutant (ENmut, H230A) L1 measured by the L1-GFP reporter assay in U2OS cells. n = 6. P-value <0.0001, each compared to RTmut using one-way ANOVA with Dunnett’s test. D. Shown is a schematic of a new GFP reporter assay modeling the recombination between a L1-cDNA intermediate and a chromosomal break to generate a chromosomal rearrangement. Left: A pCEP4-Puro plasmid contains a modified version of the classic L1-GFP reporter construct, where the 3’ UTR contains an anti-sense 5’ GFP fragment (5’GFp) that is interrupted by a sense intron (L1-5’ GFp plasmid). Transcription, splicing and retrotransposition of the L1 RNA results in a canonical insertion containing an incomplete GFP cassette and does not induce GFP expression. Nonetheless, retrotransposition of the L1 RNA generates L1 insertion intermediates containing an 5’GFP fragment with the potential to be resolved in non-canonical events. Right: A 3’ GFP fragment containing 150 bp homology to the 5’ GFP fragment from L1-5’ GFp plasmid (3’ gFP 150bp Hom) was introduced chromosomally in U2OS cells using the FRT/ Flp recombinase system (150bpHom-3’ gFP U2OS cells). A chromosomal break can be induced by the CRISRP/Cas9 system at the edge of the 150bp homology of the 3’gFP 150bp Hom sequence (Cas9/sg150bp). Hence, L1 cDNA insertion intermediates containing a 5’ GFP fragment can recombine with a broken chromosome containing a 3’ GFP fragment forming a chromosomal rearrangement and restoring GFP expression. E. Shown are representative flow cytometry plots of 150bpHom-3’ gFP U2OS cells transfected with WT or RTmut L1-5’ GFp reporter plasmid, and Cas9/ sgCTRL or Cas9/ sg150bp expressing plasmid. GFP expression is only induced with transfection of the WT L1-5’ GFp reporter plasmid and the Cas9/150bp expressing plasmid. F. Shown is the GFP frequency induced in U2OS FRT 3’ gFP (150bp Hom) cells transfected with WT, RTmut or ENmut L1-5’ GFp reporter plasmid, and Cas9/sgCTRL or Cas9/sg150bp expressing plasmid. n = 6. P-value calculated using unpaired two-tailed Student’s t tests with the Holm-Sidak correction for multiple comparisons. G. Shown is the GFP frequency induced in HEK293 150bpHom-3’ gFP cells transfected with WT, RTmut or ENmut L1-5’ GFp reporter plasmid, and Cas9/sgCTRL or Cas9/sg150bp expressing plasmid. n = 3. P-value calculated using unpaired two-tailed Student’s t tests with the Holm-Sidak correction for multiple comparisons.

Journal: bioRxiv

Article Title: L1 insertion intermediates recombine with one another or with DNA breaks to form genome rearrangements

doi: 10.1101/2025.09.17.676864

Figure Lengend Snippet: A. A working model for DNA breakage resulting from L1 retrotranspostion. L1 retrotransposition occurs via target-primed reverse transcription. L1 ORF2p can nick genomic DNA at an EN target sequence (3’-AA/TTTT-5’) to liberate a polyT sequence that complementary bind the L1 RNA polyA sequence to form a primer-template structure. L1 ORF2p can then polymerize from the free 3’OH and use the L1 RNA as a template to generate de novo L1 cDNA that can be processed into a double-stranded insertion in the genome. Alternatively, DNA breakage can occur during the resolution of the L1 cDNA intermediate into an insertion generating recombinogenic DNA break ends. B. A schematic of the classic L1-GFP reporter assay. A pCEP4-Puro plasmid constitutively expresses a native human L1 sequence (L1RP) expressing ORF1p (RNA binding protein) and ORF2p (endonuclease and reverse transcriptase) containing a GFP reporter in the 3’ UTR that marks de novo L1 insertions. The GFP cassette is in the anti-sense direction relative to L1 sequence and is interrupted by an intron in the sense direction. The continuous GFP sequence is generated when the L1 transgene is transcribed and spliced but cannot be expressed and translated until the cassette is retrotransposed. Accordingly, GFP expression marks cells with de novo L1 insertions. C. Shown is the retrotransposition frequency of WT, reverse transcriptase mutant (RTmut, D702Y) RTmut or endonuclease mutant (ENmut, H230A) L1 measured by the L1-GFP reporter assay in U2OS cells. n = 6. P-value <0.0001, each compared to RTmut using one-way ANOVA with Dunnett’s test. D. Shown is a schematic of a new GFP reporter assay modeling the recombination between a L1-cDNA intermediate and a chromosomal break to generate a chromosomal rearrangement. Left: A pCEP4-Puro plasmid contains a modified version of the classic L1-GFP reporter construct, where the 3’ UTR contains an anti-sense 5’ GFP fragment (5’GFp) that is interrupted by a sense intron (L1-5’ GFp plasmid). Transcription, splicing and retrotransposition of the L1 RNA results in a canonical insertion containing an incomplete GFP cassette and does not induce GFP expression. Nonetheless, retrotransposition of the L1 RNA generates L1 insertion intermediates containing an 5’GFP fragment with the potential to be resolved in non-canonical events. Right: A 3’ GFP fragment containing 150 bp homology to the 5’ GFP fragment from L1-5’ GFp plasmid (3’ gFP 150bp Hom) was introduced chromosomally in U2OS cells using the FRT/ Flp recombinase system (150bpHom-3’ gFP U2OS cells). A chromosomal break can be induced by the CRISRP/Cas9 system at the edge of the 150bp homology of the 3’gFP 150bp Hom sequence (Cas9/sg150bp). Hence, L1 cDNA insertion intermediates containing a 5’ GFP fragment can recombine with a broken chromosome containing a 3’ GFP fragment forming a chromosomal rearrangement and restoring GFP expression. E. Shown are representative flow cytometry plots of 150bpHom-3’ gFP U2OS cells transfected with WT or RTmut L1-5’ GFp reporter plasmid, and Cas9/ sgCTRL or Cas9/ sg150bp expressing plasmid. GFP expression is only induced with transfection of the WT L1-5’ GFp reporter plasmid and the Cas9/150bp expressing plasmid. F. Shown is the GFP frequency induced in U2OS FRT 3’ gFP (150bp Hom) cells transfected with WT, RTmut or ENmut L1-5’ GFp reporter plasmid, and Cas9/sgCTRL or Cas9/sg150bp expressing plasmid. n = 6. P-value calculated using unpaired two-tailed Student’s t tests with the Holm-Sidak correction for multiple comparisons. G. Shown is the GFP frequency induced in HEK293 150bpHom-3’ gFP cells transfected with WT, RTmut or ENmut L1-5’ GFp reporter plasmid, and Cas9/sgCTRL or Cas9/sg150bp expressing plasmid. n = 3. P-value calculated using unpaired two-tailed Student’s t tests with the Holm-Sidak correction for multiple comparisons.

Article Snippet: For this, 3’ GFP fragments were synthesized via Genewiz and cloned into the pDNA5/FRT targeting plasmid (generous gifts from Dr. Jeremy Stark).

Techniques: Reverse Transcription, Sequencing, Reporter Assay, Plasmid Preparation, Expressing, RNA Binding Assay, Generated, Mutagenesis, Modification, Construct, Flow Cytometry, Transfection, Two Tailed Test

A. GFP+ cells harbor the predicted recombination product. Shown are PCR amplification products from untransfected cells and sorted GFP+ cells derived from U2OS 150bpHom-3’ gFP cells transfected with WT L1 5’ GFp plasmid and Cas9/sg150bp expressing plasmid using primers hPGKFw (L1 5’GFp plasmid) and GFPrev (3’ gFP 150bp Hom). Amplification of GAPDH locus was used as a control. B. Schematic of the Cas9 targeted nanopore sequencing strategy to map the upstream and the downstream sequences of the GFP loci of sorted GFP+ cells. Genomic DNA was isolated from sorted GFP+ cells derived from U2OS FRT 3’ gFP (150bp Hom) cells after transfection with WT L1 5’ GFp plasmid and Cas9/sg150bp expressing plasmid. Genomic DNA was first mechanically fragmented to 30 kb length and dephosphorylated. Dephosphorylated genomic DNA was then incubated with Cas9 ribonucleoproteins using sgRNAs targeted to the 3’gFP sequence at the FRT locus. Cleaved genomic DNA ends are then ligated with Oxford Nanopore Technologies (ONT) sequencing adapters. These DNA fragments were sequenced on a MinION flow cell. Long reads were first mapped to a GFP reference to select GFP-containing long reads for subsequent mapping to the human genome (T2T) to determine their genomic integration site. C. Shown are several ONT long reads containing the 3’ GFP fragment that were mapped to the human genome (T2T) revealing the chromosomal FRT locus in U2OS cells is in chromosome 11 (7784 +). D. Shown are ONT long reads containing the 5’ GFP fragment and the recombination junction mapping to chromosome 14 (101156465 +) and showing evidence of L1 retrotransposition at the genomic site of integration: inserted at (5’-GGTT/CA-3’) and containing an polyA sequence. E. A circos plot showing the genomic locations of L1 insertion intermediates that recombined with the Cas9-mediated DNA double-strand break generated at edge of the 150 bp homology of the 3’gFP (150 bp Hom) locus in chromosome 11 to restore GFP expression via a chromosomal translocation. Each translocation is represented by an arch originating from the p arm of chromosome 11 and terminating at the location of each L1 insertion intermediate.

Journal: bioRxiv

Article Title: L1 insertion intermediates recombine with one another or with DNA breaks to form genome rearrangements

doi: 10.1101/2025.09.17.676864

Figure Lengend Snippet: A. GFP+ cells harbor the predicted recombination product. Shown are PCR amplification products from untransfected cells and sorted GFP+ cells derived from U2OS 150bpHom-3’ gFP cells transfected with WT L1 5’ GFp plasmid and Cas9/sg150bp expressing plasmid using primers hPGKFw (L1 5’GFp plasmid) and GFPrev (3’ gFP 150bp Hom). Amplification of GAPDH locus was used as a control. B. Schematic of the Cas9 targeted nanopore sequencing strategy to map the upstream and the downstream sequences of the GFP loci of sorted GFP+ cells. Genomic DNA was isolated from sorted GFP+ cells derived from U2OS FRT 3’ gFP (150bp Hom) cells after transfection with WT L1 5’ GFp plasmid and Cas9/sg150bp expressing plasmid. Genomic DNA was first mechanically fragmented to 30 kb length and dephosphorylated. Dephosphorylated genomic DNA was then incubated with Cas9 ribonucleoproteins using sgRNAs targeted to the 3’gFP sequence at the FRT locus. Cleaved genomic DNA ends are then ligated with Oxford Nanopore Technologies (ONT) sequencing adapters. These DNA fragments were sequenced on a MinION flow cell. Long reads were first mapped to a GFP reference to select GFP-containing long reads for subsequent mapping to the human genome (T2T) to determine their genomic integration site. C. Shown are several ONT long reads containing the 3’ GFP fragment that were mapped to the human genome (T2T) revealing the chromosomal FRT locus in U2OS cells is in chromosome 11 (7784 +). D. Shown are ONT long reads containing the 5’ GFP fragment and the recombination junction mapping to chromosome 14 (101156465 +) and showing evidence of L1 retrotransposition at the genomic site of integration: inserted at (5’-GGTT/CA-3’) and containing an polyA sequence. E. A circos plot showing the genomic locations of L1 insertion intermediates that recombined with the Cas9-mediated DNA double-strand break generated at edge of the 150 bp homology of the 3’gFP (150 bp Hom) locus in chromosome 11 to restore GFP expression via a chromosomal translocation. Each translocation is represented by an arch originating from the p arm of chromosome 11 and terminating at the location of each L1 insertion intermediate.

Article Snippet: For this, 3’ GFP fragments were synthesized via Genewiz and cloned into the pDNA5/FRT targeting plasmid (generous gifts from Dr. Jeremy Stark).

Techniques: Amplification, Derivative Assay, Transfection, Plasmid Preparation, Expressing, Control, Nanopore Sequencing, Isolation, Incubation, Sequencing, Generated, Translocation Assay

A . Shown is a schematic of GFP recombination reporters assaying the influence of the length of homologous sequences between a L1-cDNA intermediate and a chromosomal break for the formation of chromosomal rearrangements. Left: The L1-5’ GFp plasmid is transfected into cells to generate L1 insertion intermediates containing a 5’GFP fragment with the potential to be resolved in non-canonical events such as rearrangements. Right: 3’ GFP fragments containing varying degree of homology to the 5’ GFP fragment from L1-5’ GFp plasmid were introduced chromosomally in U2OS cells using the FRT/ Flp recombinase system: 3’gFP 150bp Hom, 3’gFP 50bp Hom, and 3’gFP 0bp Hom. Each of these 3’GFP fragments was engineered to contain a specific sgRNA target sequence to induce a Cas9-mediated chromosomal break at the edge of the homology. Hence, we can express the L1-5’GFp plasmid and the respective Cas9/sgRNA plasmid in each 3’gFP reporter cell line to monitor the influence of the homology between L1 cDNA insertion intermediates containing a 5’ GFP fragment and a broken chromosome containing a 3’ GFP fragment on the formation of a chromosomal rearrangement by assaying GFP expression. B. Shown is the GFP frequency induced in each U2OS 3’gFP reporter cell line transfected with WT, RTmut or ENmut L1-5’ GFp reporter plasmid, and the respective Cas9/sgRNA expressing plasmid or the control Cas9/sgCTRL expressing plasmid. n = 6 or 3. P-value calculated by comparing WT and ENmut versus RTmut in each condition using a two-way ANOVA with the Dunnett correction for multiple comparisons.

Journal: bioRxiv

Article Title: L1 insertion intermediates recombine with one another or with DNA breaks to form genome rearrangements

doi: 10.1101/2025.09.17.676864

Figure Lengend Snippet: A . Shown is a schematic of GFP recombination reporters assaying the influence of the length of homologous sequences between a L1-cDNA intermediate and a chromosomal break for the formation of chromosomal rearrangements. Left: The L1-5’ GFp plasmid is transfected into cells to generate L1 insertion intermediates containing a 5’GFP fragment with the potential to be resolved in non-canonical events such as rearrangements. Right: 3’ GFP fragments containing varying degree of homology to the 5’ GFP fragment from L1-5’ GFp plasmid were introduced chromosomally in U2OS cells using the FRT/ Flp recombinase system: 3’gFP 150bp Hom, 3’gFP 50bp Hom, and 3’gFP 0bp Hom. Each of these 3’GFP fragments was engineered to contain a specific sgRNA target sequence to induce a Cas9-mediated chromosomal break at the edge of the homology. Hence, we can express the L1-5’GFp plasmid and the respective Cas9/sgRNA plasmid in each 3’gFP reporter cell line to monitor the influence of the homology between L1 cDNA insertion intermediates containing a 5’ GFP fragment and a broken chromosome containing a 3’ GFP fragment on the formation of a chromosomal rearrangement by assaying GFP expression. B. Shown is the GFP frequency induced in each U2OS 3’gFP reporter cell line transfected with WT, RTmut or ENmut L1-5’ GFp reporter plasmid, and the respective Cas9/sgRNA expressing plasmid or the control Cas9/sgCTRL expressing plasmid. n = 6 or 3. P-value calculated by comparing WT and ENmut versus RTmut in each condition using a two-way ANOVA with the Dunnett correction for multiple comparisons.

Article Snippet: For this, 3’ GFP fragments were synthesized via Genewiz and cloned into the pDNA5/FRT targeting plasmid (generous gifts from Dr. Jeremy Stark).

Techniques: Plasmid Preparation, Transfection, Sequencing, Expressing, Control