CH391L/S12/Selectablegeneticmarkers: Difference between revisions

In addition to selectable genetic markers are screenable genetic markers. Screenable genetic markers function in a similar manner in that they are exogenous genes that are transformed into a cell; however, they do not confer any new sort of resistance to the cell. Instead, they cause the cell to respond differently to environmental conditions in such a way as to distinguish transformed cells from untransformed cells. This can be useful when determining the transformation efficiency of a cell, or when carefully monitoring the activity of proteins.

==Types of Selectable Markers==

===Antibiotic===

===Herbicide===

===Morphological===

===Other===

[[Image:Alternative Selective Marker.jpg|thumb|left|An alternative technique for selectable markers that avoids antibiotic resistance<cite>Parsons2011</cite>.]]

A novel approach towards selectable markers was developed in Lawrence Livermore National Laboratory, which employes a toxin/antitoxin combination of genes as a marker. The process, summarized in the figure to the left, effectively avoids the need to grow antibiotic resistant bacterial cultures on an antibiotic plate. An inducible zeta-toxin group of proteins is first introduced into an E. coli strain. A DNA strand of interest containing an zeta-antitoxin group is then transformed into the E. coli, and the entire culture is grown. The zeta-toxin group is then induced, killing off all E. coli that does not contain the antitoxin group. Besides for triggering the zeta-toxin group, no outside influence is required to select for the desired cells<cite>Parsons2011</cite>.

==Types of Screening==

===Blue/White Screening===

Blue/White Screening is commonly used in E. coli transformations. In this screening, cells are grown on agar plates in the presence of X-gal and IPTG to test for the presence of β-galactosidase enzyme. In the M15 strain of E. coli, part of the <i>lacZ</i> gene is deleted, removing the cell's ability to produce β-galactosidase. However, when transfected with a plasmid containing a <i>lacZα</i> domain, such as pUC19, the gene becomes operable and the cell produces β-galactosidase. It is possible to create a successful transformation in which β-galactosidase is not produced by inserting DNA into the <i>lacZα</i> domain. This is particularly useful to check for successful ligations. Successful ligations will not produce β-galactosidase, while unsuccessful ligations will.

X-gal, while normally colorless (i.e. white), will readily hydrolyze in the presence of β-galactosidase into a compound with a sharp blue color. Therefore, colonies with successfully transformed cells with the desired DNA will grow white, while background colonies will grow blue.  

===Green Fluorescent Protein Screening===

[[Image:Green_Fluroescent_Mice.jpg‎|thumb|left|Mice transfected with GFP. One can easily distinguish the wild-type mouse (middle) from the two mice with GFP (left and right)<cite>Moen2011</cite>.]]

In 2011, GFP was used to create an in vivo mammary model to investigate tumorigenesis in mice. Tumor cells were introduced into the mice, accompanied with GFP as a screenable marker. As the mice tumors proliferated, so did GFP. This allowed for easy differentiate between tumors and stroma cells, greatly aiding cancer researchers<cite>Moen2011</cite>.  

==Artificial Selection==

Artificial selection is a special instance in which selectable markers are often the desired gene to be introduced into a cell. For instance, rice has been transfected with a plethora of resistances using selectable markers. Glycopeptide binding protein, dihydrofolate reductase, and hygromycin phosphotransferase have all been introduced into rice, conferring resistance to bleomycin and pheomycin, methotrexate, and hygromycin B respectively. This allows farmers to use herbicides select for only rice with these markers, while eliminating the majority of invasive species<cite>Twyman2002</cite>.

==Issues==

===Genetically Modified Organisms===

Roundup Ready crops

==References==