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DNA Shuffling Examples@elegansNet - Mammalian Cellular & Viral Activities

Target Desired Trait Resolution Approach Cell Type Reference
 
HIV-1; Gag-pro- RT Enhanced replication in pig-tailed Macaque cells
  • Higher levels of replication (100 ng/ml p24) + continuous passage in pt mPBMC.
  • one out of 2.4 x 105 library members survived selection.
  • Shuffling + mutation in non-shuffled segments responsible for phenotype.
  • Single round of DNA shuffling of Gag-pro-RT segments from 11 HIV strains + selection by multiple serial passage in pt mPBMC.
  • pig-tailed Macaque cells
Pekrun K et al. J Virol. 2002 Mar;76(6):2924-35.
MLV; envelope New tropism for CHOK1 cells
  • Ability to infect CHOK1 cells.
  • One out of 1 x 106 library members surviving the selection show the phenotype.
  • Single round of DNA Shuffling of six MLV strains + selection for CHOK1 infectivity by passing on a mixture of CHO + lec8 cells.
  • Chinese Hamster Ovary cells
Soong NW et al. Molecular breeding of viruses.
Nat Genet. 2000 Aug; 25(4):436-9
.
MLV; envelope Improved stability & processing yields
  • Best clone shows no loss of titer during processing as compared to wt viruses showing 30- to 100- fold reduction.
  • A few out of 6 x 106 library members surviving the selection show the phenotype
  • Envelope of shuffled viruses are complex chimeras derived from all parents.
  • Single round of DNA Shuffling of six MLV strains + selection of stress-resistant viruses: 3 rounds of concentration + amplification.
  • mouse fibroblast cell line, 3T3
Powell SK, Nat Biotechnol. 2000 Dec; 18(12): 1279-82.

Suppl. data

Human Cytokines, Hu-IFN-as

>20 tandemly duplicated, nonallelic genes with 85-95 % aa identity

Hu-IFN-a genes are ~ 60 % identical to murine (Mu) IFN-as

Strong activity in mouse cells
  • Obtained human IFN-as more potent in mouse cells than the native mouse IFN-as .
  • Most active chimeric IFN from round 1 is derived from 6 parental IFN segments.
  • Most active chimeric IFNs from round two are derived from 5 to 6 parental segments.
  • Two rounds of DNA shuffling involving ~ 20 h-IFN-a genes and pseudogenes.
  • Chimeric IFNs expressed, purified, and screened for L929 antiviral activity (CPE-reduction) as pools of 12 or 96.
  • Active pools were deconvoluted into sequentially smaller pools to identify single active clone. Pooling strategy allowed screening of library of ~ 1600 clones with 68 antiviral assays.
  • Most active chimera from a 1st round library of ~1500 clones is 87-fold more active than the best of the Hu-IFN-as.
  • In the 2nd round, pooled and pair-wise mating results involving ~1000 individual clones compared. The most active clones resulted from pair-wise mating. Their activity far exceeds those of the most active non-shuffled h-IFN-as and 2- to 3-fold higher than Mu-IFN-as.
  • L929 (Mouse fibroblast)
  • Daudi (Human B lymphoblast)
  • CHO cells
Chang CC et al. Nat Biotechnol. 1999 Aug; 17(8):793-7.
 

Created 01/12/02

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Updated: 05/05/2002

 

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