Transfection protoplast

Protocols for protoplast transfection and particle bombardment of leaf epidermal cells... 

Most protocols for the isolation of protoplasts from Cowpea, Petunia, or Tobacco mesophyll cells are based on [93, 94] and others [95-97], After the isolation of protoplasts from the cell type of choice, the transfection is carried out as follows (based on [51, 96]) ... 

Finally, we tested the particle bombardment method, a biolistic approach that was shown before to work when other approaches have failed. In particular, particle bombardment has allowed the generation of transgenic plants such as crop species, which are not susceptible to Agrobacterium tumefaciens or cannot be regenerated from protoplasts. Furthermore, particle bombardment has facilitated organelle transformation in intact cells and the genetic modification of cultured cells that were not accessible to other transfection techniques. 

Transfection, DNA uptake in eukaryotic systems, often is more problematic then bacterial transformation the mode of DNA uptake is poorly understood and efficiency is much lower. In yeast, cell walls can be digested with degradative enzymes to yield fragile protoplasts, which are then able to take up DNA. Cell walls are resynthesized after removal of the degrading enzymes. Mammalian cells take up DNA after precipitation onto their surface with calcium phosphate [Fugene 6 (Roche) Lipofectin (Life Technologies) Effectene (Qiagen)]. Electroporation is often more efficient for transfection in eukaryotic cell systems, especially in yeasts. 

Recently, a photoactivatable variant from Aequoria victoria green fluorescent protein (pa-GFP) was reported (Patterson and Lippincott-Schwartz 2002), yielding an increase in fluorescence emission intensity (at k 520 nm) by a factor of 100 when excited at k 488 nm after spectral activation at A. 408 nm. This phenomenon is due to an internal photoconversion process in the protein and allows spectral photoactivation of this protein in a very local way such as in the nucleus of a living cell (Post et al. 2005). In tobacco BY-2 protoplasts, we transiently co-expressed pa-GFP or pa-GFP fusion proteins and red-fluorescent protein (DsRed)-tagged prenylated Rab acceptor 1 (Pral At2g38360), a membrane protein that localizes in speckles around the nuclear envelope. The DsRed transfection allows proper cell identification and visualization before activation (via Pral -DsRed fluorescence). After pa-GFP... 

On the other hand, translocation of the MYB transcription factor LCLl fused to pa-GFP from the nucleus to the cytoplasm and its intracellular localization dynamics depends on facilitated nuclear export versus facilitated nuclear import. Protoplasts co-transfected with At2g38360-DsRed and pa-GFP-LCLl were subjected to the 2P-activation procedure and the decrease of nuclear fluorescence intensity was monitored... 

The firefly gene has also been used to identify promoter elements of the cauliflower mosaic virus (CaMV) 35S RNA promoter. Protoplasts from a Daucus carota (carrot) cell line were transfected with various DNAs with a modified electroporation tech-... 

While these approaches represent many of the most common transfection techniques, this is certainly not an exhaustive list. Other methods, such as microinjection and protoplast fusion not discussed here, are also currently available. 

---