Transfection, identification

Oh S-W et al. Identification of differentially expressed genes in human hepatoblastoma cell line (HepG2] and HBV-X transfected hepatobalstoma cell line (HepG2-4X). Col Cells 1998 8 212-218. 

Ex vivo systems derived from animals and from human organs can be used to investigate the in vitro metabolism of xenobiotics. Cell lines, which are transfected to express species-specific metabolic enzymes, can also be used to identify the enzymes involved in the metabolism of a specific substance. Blocking the metabolism by an enzyme specific substrate or by antibodies is also helpful for the identification of the enzymes involved in the metabolism of a substance. 

Identification of this gene rearrangement is the sine qua non for the diagnosis of CML, and its presence in the hematopoietic cells of virtually all patients with CML made it an ideal target for the development of therapeutic agents. Proof that BCR-ABL 1 was crucial for the development of CML was shown when the hybrid gene was transfected into bone marrow cells of mice which were subsequently transplanted into an irradiated syngeneic recipient and shown to lead to the development of a CML-like syndrome (5). 

Figure 15.6 Schematic depiction of one example of protein identification by mass spectrometry. Genes of interest are tagged, then transfected into mammalian cells, and proteins associated with the cognate tagged protein are purified by affinity methods. Separation of the complex is carried out by 2D SDS-PAGE. Identification of the proteins is by MALDI and ESI (Blackwell, 1999).

Finally, Ruthardt andBraeuchle summarize recent findings, describing transfection pathways of non-viral gene carriers by single particle tracking approaches. This approach allows the detailed identification of potential hurdles for efficient nucleic acid delivery from a single cell viewpoint. 

It should be noted that the band we identify as dysbindin-1C does not correspond to the band of the same name recognized by the dysbindin-1 antibody of Oyama et al. (2009). In COS-7 cells transfected to express human dysbindin-1C, their antibody recognizes two proteins. They interpret the upper band as dysbindin-1C and the lower one as a degradation product. This identification requires further confirmation, because the upper band has a molecular mass greater than that for dysbindin-lB, which is 100 aa longer than dysbindin-1C. 

The power of the screening or selection method ultimately delineates the extent to which the sequence space can be explored. Screening usually involves the physical separation of mutants identified on some phenotypic change such as colony size or color. It is essentially a brute force approach that is amenable to amplification by robotics and is limited to identification of mutants in a population of transfected bacteria or cells totaling at most one million and more with typically only a few thousand clones each harboring a different mutant gene. Selection takes advantage... 

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... 

Ji, C., Li, L., Gebre, M., Pasdar, M. and Li, L. (2005) Identification and quantitation of differentially expressed proteins in E-cadherin deficient SCC9 cells and SCC9 transfectants expressing E-cadherin by dimethyl isotope labeling, LC-MALDI MS and MS/MS. J. Proteome Res. 4, 1419-1426. 

---