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Algae transformation methods -
References:
Review. Methodologies for…
Algae transformation methods -
References:
- Review. Methodologies for transferring DNA into eukaryotic microalgae
Challenges
Cell wall
All of the eukaryotic microalgae transformed have a cell wall, an organelle that resists efforts to achieve DNA penetration during transformation experiments. Cell walls vary from the more elastic and thinner type of Chlamydomonas, Volvox or Dunaliella, to the highly gelatinous type of red algae, the more rigid type of Chlorella, and the extremely hard siliceous type possessed by diatoms
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Cell size and shape
"The shape and size of microalgal cells are also important variables to take into account during transfection procedures. Extreme transfection conditions are required when the smaller (<5 µm) Chlorella and diatoms are to be transformed, while the larger (10- 50 µm) Chlamydomonas, Dunaliella, Haematococcus and Euglena, etc. are generally less demanding."
Haploid and diploid
Haploidy (as shown by Chlamydomonas and Volvox) is considered an advantage when trying to obtain transformants since this condition allows the imme- diate phenotypic expression of non-lethal mutations. Diploidy, in contrast, is a problem that has to be circumvented. However, most of the microalgae that have been successfully transformed are diploid or alternate between haploid and diploid stages during their cell cycles.
Methods
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Electroporation
In electroporation, electric pulses are varied in
intensity, duration and number to control the efficiency of exogenous DNA delivery.
Preincubation, application of the electric pulse and postincubation are preferen- tially performed at ~ 4ºC to enhance survival and to allow time for the exogenous DNA to enter the cell before the complete resealing of the electrically- induced holes (Chu et al., 1987).
The medium used for electroporation is important since: i) its resistance and osmolarity influence the pulse duration, which in turn influences cell survival, ii) the concentration of divalent ions affects DNA structure and membrane stability, and iii) its general composition affects cell survival since some medium penetrates the electrically permeabilized cells.
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