9/21/2023 0 Comments Dna shuffling r studio library![]() Bioluminescent images were obtained at the end of 1 month using a Xenogen IVIS system. ( a) Luciferase transgene expression in the hindlimbs of Balb/C mouse injected with 10 10 vector genomes of the novel adeno-associated virus (AAV) variant and parental AAV1, 2. ( b) Dot blot demonstrating the directed evolution of cell type–specific AAV variant from CS1 cells after five infectious cycles ( n = 5). Subsequent steps will involve development of novel AAV variants as targeted vectors for in vivo gene delivery applications. Specific cell line (hamster melanoma CS1 cells) previously shown to be restricted to transduction of AAVs will be tested with the chimeric AAV library for a premissive clone. AAV particle library was generated by plasmid transfection protocol. Following this reassembly reaction, PCR amplification with specific primers was used to generate full-length Cap chimeras for cloning into wild-type AAV2 backbone to generate shuffled plasmid library. AAV Cap genes were randomly fragmented and reassembled using cycles of denaturation, annealing, and extension using PCR. ( a) Strategy for generating a combinatorial adeno-associated virus (AAV) library that will be subjected to directed evolution to isolate cell type/receptor-specific AAV mutants. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Capsid genomes of adeno-associated virus (AAV) serotypes 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. doi: 10.We report a DNA shuffling-based approach for developing cell type-specific vectors through directed evolution. He L, Friedman AM, Bailey-Kellogg C (2012) Algorithms for optimizing cross-overs in DNA shuffling.Moore GL, Maranas CD (2002) eCodonOpt: a systematic computational framework for optimizing codon usage in directed evolution experiments.Moore GL, Maranas CD, Lutz S, Benkovic SJ (2001) Predicting crossover generation in DNA shuffling.Moore GL, Maranas CD (2000) Modeling DNA mutation and recombination for directed evolution experiments.O’Maille PE, Bakhtina M, Tsai MD (2002) Structure-based combinatorial protein engineering (SCOPE).Coco WM, Levinson WE, Crist MJ, Hektor HJ, Darzins A, Pienkos PT, Squires CH, Monticello DJ (2001) DNA shuffling method for generating highly recombined genes and evolved enzymes.Zhao H, Giver L, Shao Z, Affholter JA, Arnold FH (1998) Molecular evolution by staggered extension process (StEP) in vitro recombination.Zhang YX, Perry K, Vinci VA, Powell K, Stemmer WP, del Cardayre SB (2002) Genome shuffling leads to rapid phenotypic improvement in bacteria.Soong NW, Nomura L, Pekrun K, Reed M, Sheppard L, Dawes G, Stemmer WP (2000) Molecular breeding of viruses.Powell SK, Kaloss MA, Pinkstaff A, McKee R, Burimski I, Pensiero M, Otto E, Stemmer WP, Soong NW (2000) Breeding of retroviruses by DNA shuffling for improved stability and processing yields.Patten PA, Howard RJ, Stemmer WP (1997) Applications of DNA shuffling to pharmaceuticals and vaccines.Crameri A, Raillard SA, Bermudez E, Stemmer WP (1998) DNA shuffling of a family of genes from diverse species accelerates directed evolution.Chang CC, Chen TT, Cox BW, Dawes GN, Stemmer WP, Punnonen J, Patten PA (1999) Evolution of a cytokine using DNA family shuffling.Stemmer WP (1994) DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |