Skeletal muscle satellite tv for pc cells (SCs) are stem cells chargeable for muscle improvement and regeneration. Though CRISPR/Cas9 has been broadly used, its utility in endogenous SCs stays elusive. Right here, we generate mice expressing Cas9 in SCs and obtain sturdy enhancing in juvenile SCs on the postnatal stage via AAV9-mediated brief information RNA (sgRNA) supply.
Moreover, we reveal that quiescent SCs are immune to CRISPR/Cas9-mediated enhancing. As a proof of idea, we show environment friendly enhancing of grasp transcription issue (TF) Myod1 locus utilizing the CRISPR/Cas9/AAV9-sgRNA system in juvenile SCs.
Software on two key TFs, MYC and BCL6, unveils distinct capabilities in SC activation and muscle regeneration. Notably, we reveal that MYC orchestrates SC activation via regulating 3D genome structure. Its depletion ends in strengthening of the topologically associating area boundaries thus might have an effect on gene expression.
Altogether, our examine establishes a platform for enhancing endogenous SCs that may be harnessed to elucidate the performance of key regulators governing SC actions.
Oleuropein Prompts Neonatal Neocortical Proteasomes, however Proteasome Gene Concentrating on by AAV9 Is Variable in a Clinically Related Piglet Mannequin of Mind Hypoxia-Ischemia and Hypothermia
Cerebral hypoxia-ischemia (HI) compromises the proteasome in a clinically related neonatal piglet mannequin. Defending and activating proteasomes may very well be an adjunct remedy to hypothermia. We investigated whether or not chymotrypsin-like proteasome exercise differs regionally and developmentally within the neonatal mind.
We additionally examined whether or not neonatal mind proteasomes will be modulated by oleuropein, an experimental pleiotropic neuroprotective drug, or by concentrating on a proteasome subunit gene utilizing recombinant adeno-associated virus-9 (AAV).
Throughout post-HI hypothermia, we handled piglets with oleuropein, used AAV-short hairpin RNA (shRNA) to knock down proteasome activator 28γ (PA28γ), or enforced PA28γ utilizing AAV-PA28γ with inexperienced fluorescent protein (GFP).
Neonatal neocortex and subcortical white matter had higher proteasome exercise than did liver and kidney. Neonatal white matter had increased proteasome exercise than did juvenile white matter. Decrease arterial pH 1 h after HI correlated with higher subsequent cortical proteasome exercise.
With rising mind homogenate protein enter into the assay, the preliminary proteasome exercise elevated solely amongst shams, whereas HI elevated complete kinetic proteasome exercise. OLE elevated the preliminary neocortical proteasome exercise after hypothermia.
AAV drove GFP expression, and white matter PA28γ ranges correlated with proteasome exercise and subunit ranges. Nevertheless, AAV proteasome modulation diverse. Thus, neonatal neocortical proteasomes will be pharmacologically activated.
HI slows the preliminary proteasome efficiency, however then augments ongoing catalytic exercise. AAV-mediated genetic manipulation within the piglet mind holds promise, although proteasome gene concentrating on requires additional improvement.
AAV9 Structural Rearrangements Induced by Endosomal Trafficking pH and Glycan Attachment
Adeno-associated viruses (AAVs) are small non-enveloped ssDNA viruses, which might be at present being developed as gene remedy biologics. After cell entry, AAVs site visitors to the nucleus utilizing the endo-lysosomal pathway.
The next lower in pH triggers conformational adjustments to the capsid that allows the externalization of the capsid protein (VP) N-termini, together with the distinctive area of the minor capsid protein VP1 (VP1u), which allows phospholipase exercise required for the capsid lysosomal egress.
Right here, we report the AAV9 capsid construction, decided on the endosomal pHs (7.4, 6.0, 5.5, and 4.0) and terminal galactose-bound AAV9 capsids at pHs 7.Four and 5.5 utilizing cryo-electron microscopy and three-dimensional picture reconstruction.
Taken collectively these research present perception into AAV9 capsid conformational adjustments on the 5-fold pore throughout endosomal trafficking, each within the presence and absence of its mobile glycan receptor.
We visualized, for the primary time, that acidification induces the externalization of the VP3 and probably VP2 N-termini, presumably in prelude to the externalization of VP1u at pH 4.0, that’s important for lysosomal membrane disruption. As well as, the structural examine of AAV9-galactose interactions demonstrates AAV9 stays connected to its glycan receptor on the late endosome pH 5.5.
This interplay considerably alters the conformational stability of the variable area I of the VPs, in addition to the dynamics related to VP N-terminus externalization. Significance There are 13 distinct Adeno-associated virus (AAV) serotypes which might be structurally homologous and whose capsid proteins (VP1-3) are related in amino acid sequence.
Nevertheless, AAV9 is without doubt one of the mostly studied and used as gene remedy vector. That is half as a result of, AAV9 is able to crossing the blood mind barrier in addition to readily transduces a big selection of tissues, together with the central nervous system.
On this examine we offer AAV9 capsid structural perception throughout intracellular trafficking. Though the AAV capsid has been proven to externalize the N-termini of its VPs, to enzymatically disrupt the lysosome membrane at low pH, there was no structural proof to verify this.
By using AAV9 as our mannequin, we offer the primary structural proof that the externalization course of happens on the protein interface on the icosahedral 5-fold symmetry axis and will be triggered by reducing pH.
The AAV9 variant capsid AAV-F mediates widespread transgene expression in non-human primate spinal wire after intrathecal administration
Intrathecal supply of AAV9 into the subarachnoid area has been proven to transduce spinal wire and mind and be much less affected by pre-existing antibodies, that are decrease in cerebral spinal fluid (CSF). Nonetheless, effectivity of transduction must be improved.
Lately we recognized a brand new capsid from a library choice in mice, referred to as AAV-F, that allowed sturdy transduction of the spinal wire grey matter after lumbar injection. Right here we take a look at transduction of spinal wire by AAV-F (n=3) in comparison with AAV9 (n=2), utilizing a reporter gene, in cynomolgus monkeys after lumbar intrathecal injection.
Utilizing an automatic picture evaluation method to sensitively quantitate reporter gene expression in spinal wire, we discovered that AAV-F capsid mediated barely increased transgene expression (each in percentages of cells and depth of immunostaining) in motor neurons and interneurons, within the lumbar and thoracic areas, in comparison with AAV9.
Curiously, though AAV-F mediated increased transgene expression in spinal wire, the variety of genomes in spinal wire and periphery have been on common decrease for AAV-F than AAV9, which counsel decrease numbers of genomes have been in a position to mediate increased transgene expression in spinal wire with this capsid.
In distinction DRG transduction effectivity was decrease for AAV-F in comparison with AAV9 on common. Curiously, we additionally noticed transduction of Schwann cells in sciatic nerve in two NHPs injected with AAV-F however none with AAV9.
AAV-293 Cell Line |
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CL-0019 | Elabscience Biotech | 1×10⁶ cells/vial | EUR 420 |
Description: Homo sapiens, Human |
AAV-293 Cells Complete Medium |
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CM-0019-125mL4 | Elabscience Biotech | 125 mL×4 | EUR 98 |
Description: Complete Growth Medium |
AAV1 SaCas9 |
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78479 | BPS Bioscience | 50 µl x 2 | EUR 545 |
Description: Adeno-Associated Virus Serotype 1 (AAV1) exhibits high homology with other AAV serotypes. AAV1 efficiently transduces muscle tissue, as determined by a region of the capsid protein VP1 (amino acids 350 to 430) which functions as a major determinant of tissue tropism._x000D_Cas9 is an endonuclease enzyme that introduces a double stranded break into the DNA when recruited to a specific DNA sequence by the sgRNA (single guide RNA). This double stranded break is repaired in mammalian cells either through Non-Homologous End Joining or Homologous Recombination. Non-Homologous End Joining often results in the deletion or insertion of several base pairs at the cut site, which, when resulting in a frameshift, causes the functional inactivation of the gene._x000D_SaCas9 (Staphylococcus aureus CRISPR-associated protein 9) has demonstrated high cutting efficiency in mammalian cells, and its smaller size makes it ideal for packaging into AAV. SaCas9 recognizes a longer protospacer adjacent motif (PAM) site, 5'-NNGRRT-3', than the more traditional SpCas9 (Streptococcus pyogenes CRISPR-associated protein 9). These AAV particles constitutively express SaCas9 under the control of a CMV promoter. |
AAV2 SaCas9 |
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78480 | BPS Bioscience | 50 µl x 2 | EUR 545 |
Description: Adeno-Associated Virus serotype 2 (AAV2) is the best characterized AAV serotype. Nearly all recombinant AAV serotypes utilize the AAV2 inverted terminal repeats (ITRs). AAV2 requires the expression of Heparan Sulfate Proteoglycan (HSPG) on the surface of host for cells for binding and internalization. Of nearly all the discovered AAV serotypes, AAV2 has the best transduction efficiency in cell culture and is the best tool for in vitro studies._x000D_Cas9 is an endonuclease enzyme that introduces a double stranded break into the DNA when recruited to a specific DNA sequence by the sgRNA (single guide RNA). This double stranded break is repaired in mammalian cells either through Non-Homologous End Joining or Homologous Recombination. Non-Homologous End Joining often results in the deletion or insertion of several base pairs at the cut site, which, when resulting in a frameshift, causes the functional inactivation of the gene._x000D_SaCas9 (Staphylococcus aureus CRISPR-associated protein 9) has demonstrated high cutting efficiency in mammalian cells, and its smaller size makes it ideal for packaging into AAV. SaCas9 recognizes a longer protospacer adjacent motif (PAM) site, 5'-NNGRRT-3', than the more traditional SpCas9 (Streptococcus pyogenes CRISPR-associated protein 9). These AAV particles constitutively express SaCas9 under the control of a CMV promoter. The protein contains a C-terminus HA tag. |
AAV3 SaCas9 |
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78481 | BPS Bioscience | 50 µl x 2 | EUR 545 |
Description: Adeno-Associated Virus Serotype 3 (AAV3) shares 82% sequence homology with AAV2, and like AAV2, requires the Heparan Sulfate Proteoglycan (HSPG) receptor for cell attachment. AAV3 vectors transduce human liver cancer cells extremely efficiently because AAV3 utilizes the human Hepatocyte Growth Factor Receptor (hHGFR) as a co-receptor for viral entry, which is highly expressed in these cells. Both the extracellular and intracellular kinase domains of hHGFR are required for AAV3-mediated transgene expression._x000D_Cas9 is an endonuclease enzyme that introduces a double stranded break into the DNA when recruited to a specific DNA sequence by the sgRNA (single guide RNA). This double stranded break is repaired in mammalian cells either through Non-Homologous End Joining or Homologous Recombination. Non-Homologous End Joining often results in the deletion or insertion of several base pairs at the cut site, which, when resulting in a frameshift, causes the functional inactivation of the gene._x000D_SaCas9 (Staphylococcus aureus CRISPR-associated protein 9) has demonstrated high cutting efficiency in mammalian cells, and its smaller size makes it ideal for packaging into AAV. SaCas9 recognizes a longer protospacer adjacent motif (PAM) site, 5'-NNGRRT-3', than the more traditional SpCas9 (Streptococcus pyogenes CRISPR-associated protein 9). These AAV particles constitutively express SaCas9 under the control of a CMV promoter. |
AAV5 SaCas9 |
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78483 | BPS Bioscience | 50 µl x 2 | EUR 545 |
Description: Adeno-Associated Virus Serotype 5 (AAV5) differs from other parvovirus serotypes according to serological and DNA hybridization data, and AAV5 also uses different inverted terminal repeats (ITRs) compared to other AAV serotypes. AAV5 is the most efficient vector for transducing sensory neurons and is good at mediating gene transfer into human and murine airway epithelia. In addition, AAV5 vectors show a higher tropism for both mouse and human dendritic cells than AAV1, AAV2, AAV7, or AAV8._x000D_Cas9 is an endonuclease enzyme that introduces a double stranded break into the DNA when recruited to a specific DNA sequence by the sgRNA (single guide RNA). This double stranded break is repaired in mammalian cells either through Non-Homologous End Joining or Homologous Recombination. Non-Homologous End Joining often results in the deletion or insertion of several base pairs at the cut site, which, when resulting in a frameshift, causes the functional inactivation of the gene._x000D_SaCas9 (Staphylococcus aureus CRISPR-associated protein 9) has demonstrated high cutting efficiency in mammalian cells, and its smaller size makes it ideal for packaging into AAV. SaCas9 recognizes a longer protospacer adjacent motif (PAM) site, 5'-NNGRRT-3', than the more traditional SpCas9 (Streptococcus pyogenes CRISPR-associated protein 9). These AAV particles constitutively express SaCas9 under the control of a CMV promoter. |
AAV6 SaCas9 |
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78484 | BPS Bioscience | 50 µl x 2 | EUR 545 |
Description: Adeno-Associated Virus serotype 6 (AAV6) appears to be related to AAV1 by sequence analysis and shows the best transduction efficiency in pancreatic beta-cells compared to other AAV serotypes. AAV6 vectors are also particularly effective in the transduction of human prostate, breast, and liver cancer cells._x000D_Cas9 is an endonuclease enzyme that introduces a double stranded break into the DNA when recruited to a specific DNA sequence by the sgRNA (single guide RNA). This double stranded break is repaired in mammalian cells either through Non-Homologous End Joining or Homologous Recombination. Non-Homologous End Joining often results in the deletion or insertion of several base pairs at the cut site, which, when resulting in a frameshift, causes the functional inactivation of the gene._x000D_SaCas9 (Staphylococcus aureus CRISPR-associated protein 9) has demonstrated high cutting efficiency in mammalian cells, and its smaller size makes it ideal for packaging into AAV. SaCas9 recognizes a longer protospacer adjacent motif (PAM) site, 5'-NNGRRT-3', than the more traditional SpCas9 (Streptococcus pyogenes CRISPR-associated protein 9). These AAV particles constitutively express SaCas9 under the control of a CMV promoter. |
AAV8 SaCas9 |
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78485 | BPS Bioscience | 50 µl x 2 | EUR 545 |
Description: Adeno-Associated Virus serotype 8 (AAV8) was isolated from rhesus monkey tissue, and the AAV8 rep and cap nucleotide sequences have 88% homology with AAV7 and 82% with AAV2. AAV8 exhibits greater transduction efficiency in the liver than other AAV serotypes. AAV8 and 9 have recently been used to correct disease-causing mutations and improve muscle function in mouse models of Duchenne muscular dystrophy._x000D_Cas9 is an endonuclease enzyme that introduces a double stranded break into the DNA when recruited to a specific DNA sequence by the sgRNA (single guide RNA). This double stranded break is repaired in mammalian cells either through Non-Homologous End Joining or Homologous Recombination. Non-Homologous End Joining often results in the deletion or insertion of several base pairs at the cut site, which, when resulting in a frameshift, causes the functional inactivation of the gene._x000D_SaCas9 (Staphylococcus aureus CRISPR-associated protein 9) has demonstrated high cutting efficiency in mammalian cells, and its smaller size makes it ideal for packaging into AAV. SaCas9 recognizes a longer protospacer adjacent motif (PAM) site, 5'-NNGRRT-3', than the more traditional SpCas9 (Streptococcus pyogenes CRISPR-associated protein 9). These AAV particles constitutively express SaCas9 under the control of a CMV promoter. |
AAV1 ELISA Kit |
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55R-PROPRAAV1 | Fitzgerald | 96 tests | EUR 1406.4 |
Description: ELISA kit for detection of AAV1 in the research laboratory |
AAV5 ELISA Kit |
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55R-PROPRAAV5 | Fitzgerald | 96 tests | EUR 1442.4 |
Description: ELISA kit for detection of AAV5 in the research laboratory |
AAV1 ZsGreen |
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78443 | BPS Bioscience | 50 µl x 2 | EUR 545 |
Description: Adeno-Associated Virus Serotype 1 (AAV1) exhibits high homology with other AAV serotypes. AAV1 efficiently transduces muscle tissue, as determined by a region of the capsid protein VP1 (amino acids 350 to 430) which functions as a major determinant of tissue tropism._x000D_These AAV1 particles constitutively express ZsGreen under a CMV promoter. ZsGreen is a human codon-optimized variant of the green fluorescent protein isolated from reef coral (Zoanthus sp). It has been engineered for higher expression in mammalian cells and is up to four times brighter than enhanced GFP (eGFP). ZsGreen expression and AAV1 transduction efficiency can easily be verified and optimized by fluorescence microscopy or flow cytometry. ZsGreen has an excitation wavelength of 493 nm and an emission wavelength of 505 nm. |
AAV2 ZsGreen |
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78444 | BPS Bioscience | 50 µl x 2 | EUR 545 |
Description: Adeno-Associated Virus Serotype 2 (AAV2) is the best characterized AAV serotype. Nearly all recombinant AAV serotypes utilize the AAV2 inverted terminal repeats (ITRs). AAV2 requires the expression of Heparan Sulfate Proteoglycan (HSPG) on the surface of host cells for cell binding and internalization. Of nearly all the discovered AAV serotypes, AAV2 has the best transduction efficiency in cell culture and is the best tool for in vitro studies._x000D_These AAV2 particles constitutively express ZsGreen under a CMV promoter. ZsGreen is a human codon-optimized variant of the green fluorescent protein isolated from reef coral (Zoanthus sp). It has been engineered for higher expression in mammalian cells and is up to four times brighter than enhanced GFP (eGFP). ZsGreen expression and transduction efficiency can easily be verified and optimized by fluorescence microscopy or flow cytometry. ZsGreen has an excitation wavelength of 493 nm and an emission wavelength of 505 nm. |
AAV3 ZsGreen |
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78445 | BPS Bioscience | 50 µl x 2 | EUR 545 |
Description: Adeno-Associated Virus Serotype 3 (AAV3) shares 82% sequence homology with AAV2, and like AAV2, requires Heparan Sulfate Proteoglycan (HSPG) for cellular attachment. AAV3 vectors transduce human liver cancer cells extremely efficiently because AAV3 utilizes the human Hepatocyte Growth Factor Receptor (hHGFR) as a co-receptor for viral entry, which is highly expressed in these cells. Both the extracellular domain and the intracellular kinase domain of hHGFR are required for AAV3-mediated transgene expression._x000D_These AAV3 particles constitutively express ZsGreen under a CMV promoter. ZsGreen is a human codon-optimized variant of the green fluorescent protein isolated from reef coral (Zoanthus sp). It has been engineered for higher expression in mammalian cells and is up to four times brighter than enhanced GFP (eGFP). ZsGreen expression and AAV3 transduction efficiency can easily be verified and optimized by fluorescence microscopy or flow cytometry. ZsGreen has an excitation wavelength of 493 nm and an emission wavelength of 505 nm. |
AAV4 ZsGreen |
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78446 | BPS Bioscience | 50 µl x 2 | EUR 545 |
Description: Adeno-Associated Virus Serotype 4 (AAV4) contains an expanded p5 promoter region compared to either AAV2 or AAV3. The AAV4 Rep gene product shows greater than 90% homology with the Rep products of AAV2 and AAV3. AAV4 can transduce human, monkey, and rat cells, and efficiently transduces type B astrocytes in the subventricular zone and glia overlying the rostral migratory stream neural tube._x000D_These AAV particles constitutively express ZsGreen under a CMV promoter. ZsGreen is a human codon-optimized variant of the green fluorescent protein isolated from reef coral (Zoanthus sp). It has been engineered for higher expression in mammalian cells and is up to four times brighter than enhanced GFP (eGFP). ZsGreen expression and AAV transduction efficiency can easily be verified and optimized by fluorescence microscopy or flow cytometry. ZsGreen has an excitation wavelength of 493 nm and an emission wavelength of 505 nm. |
AAV5 ZsGreen |
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78447 | BPS Bioscience | 50 µl x 2 | EUR 545 |
Description: Adeno-Associated Virus Serotype 5 (AAV5) differs from other parvovirus serotypes according to serological and DNA hybridization data, and AAV5 also uses different inverted terminal repeats (ITRs) compared to other AAV serotypes. AAV5 is the most efficient vector for transducing sensory neurons and is good at mediating gene transfer to human and murine airway epithelia. In addition, AAV5 vectors show a higher tropism for mouse and human dendritic cells than AAV1, AAV2, AAV7, or AAV8._x000D_These AAV5 particles constitutively express ZsGreen under a CMV promoter. ZsGreen is a human codon-optimized variant of the green fluorescent protein isolated from reef coral (Zoanthus sp). It has been engineered for higher expression in mammalian cells and is up to four times brighter than enhanced GFP (eGFP). ZsGreen expression and transduction efficiency can easily be verified and optimized by fluorescence microscopy or flow cytometry. ZsGreen has an excitation wavelength of 493 nm and an emission wavelength of 505 nm. |
AAV6 ZsGreen |
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78448 | BPS Bioscience | 50 µl x 2 | EUR 545 |
Description: Adeno-Associated Virus Serotype 6 (AAV6) appears to be related to AAV1 by sequence analysis and shows the best transduction efficiency in pancreatic beta-cells._x000D_These AAV6 particles constitutively express ZsGreen under a CMV promoter. ZsGreen is a human codon-optimized variant of the green fluorescent protein isolated from reef coral (Zoanthus sp). It has been engineered for higher expression in mammalian cells and is up to four times brighter than enhanced GFP (eGFP). ZsGreen expression and AAV6 transduction efficiency can easily be verified and optimized by fluorescence microscopy or flow cytometry. ZsGreen has an excitation wavelength of 493 nm and an emission wavelength of 505 nm. |
AAV8 ZsGreen |
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78449 | BPS Bioscience | 50 µl x 2 | EUR 545 |
Description: Adeno-Associated Virus Serotype 8 (AAV8) was first isolated from rhesus monkey tissue, and the AAV8 rep and cap nucleotide sequences have 88% homology with AAV7 and 82% with AAV2. AAV8 exhibits greater transduction efficiency in the liver than other AAV serotypes._x000D_These AAV8 particles constitutively express ZsGreen under a CMV promoter. ZsGreen is a human codon-optimized variant of the green fluorescent protein isolated from reef coral (Zoanthus sp). It has been engineered for higher expression in mammalian cells and is up to four times brighter than enhanced GFP (eGFP). ZsGreen expression and transduction efficiency can easily be verified and optimized by fluorescence microscopy or flow cytometry. ZsGreen has an excitation wavelength of 493 nm and an emission wavelength of 505 nm. |
AAV4 antibody |
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10R-2443 | Fitzgerald | 50 ug | EUR 699.6 |
Description: Mouse monoclonal AAV 4 antibody |
AAV5 antibody |
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10R-2444 | Fitzgerald | 50 ug | EUR 699.6 |
Description: Mouse monoclonal AAV 5 antibody |
AAV4 antibody |
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10R-2470 | Fitzgerald | 5 mL | EUR 486 |
Description: Mouse monoclonal AAV 4 antibody |
AAV5 antibody |
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10R-2471 | Fitzgerald | 5 mL | EUR 486 |
Description: Mouse monoclonal AAV 5 antibody |
AAV5 antibody |
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10R-2472 | Fitzgerald | 5 mL | EUR 486 |
Description: Mouse monoclonal AAV 5 antibody |
AAV1 antibody |
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10R-2473 | Fitzgerald | 5 mL | EUR 550.8 |
Description: Mouse monoclonal AAV1 antibody |
AAV6 antibody |
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10R-2480 | Fitzgerald | 5 mL | EUR 549.6 |
Description: Mouse monoclonal AAV6 antibody |
AAV8 antibody |
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10R-2481 | Fitzgerald | 5 mL | EUR 553.2 |
Description: Mouse monoclonal AAV8 antibody |
AAV8/9 antibody |
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10R-2482 | Fitzgerald | 5 mL | EUR 549.6 |
Description: Mouse monoclonal AAV8/9 antibody |
AAV8 antibody |
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10R-3126 | Fitzgerald | 100 uL | EUR 974.4 |
Description: Mouse monoclonal AAV8 antibody |
AAV2 antibody |
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10R-A111a | Fitzgerald | 50 ug | EUR 706.8 |
Description: Mouse monoclonal AAV2 antibody |
AAV2 antibody |
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10R-A140a | Fitzgerald | 50 ug | EUR 818.4 |
Description: Mouse monoclonal AAV2 antibody |
General, our information demonstrates the utility of automated picture evaluation for quantitation of AAV transduction within the spinal wire and the favorable on course:off track transduction profile means that the AAV-F capsid be thought-about for gene remedy purposes centered on treating the spinal wire after intrathecal supply.