Generation of Tagged Bestrophin Proteins by Recombineering

Tiffani Cherry
Marquette University
Mentor:  Dr. Edward Blumenthal

Bestrophins are a newly identified family of genes that are believed to encode calcium activated chloride channels.  Bestrophins are expressed in the Malpighian tubules of Drosophila melanogaster.   Malpighian tubules are one component of the insect excretory system.  My research has focused on finding the location of two of the bestrophin proteins – dbest1 and dbest2.  The technique used to tag these proteins is recombineering.  Recombineering is recombination-mediated genetic engineering.  A GFP tag and a HA tag are used to tag these proteins.  Progress toward finding where the bestrophins are localized has been made.  Thus far the BACs containing dbest1 and dbest2 have been moved into the bacterial strain SW102.  SW102 is a strain of bacteria in which homologous recombination is very efficient when heat induced.  PCR was done to confirm that the BACs were moved into SW102.  A galK insert used for selection has been inserted into dbest1 and dbest2 by recombineering.  Recombineering has been done on dbest2 using the HA tag.  Colonies have been obtained from this replacement of galK with the HA tag.  These colonies will be tested for the presence of the HA tag by PCR.  The next step is to replace the galK insert in dbest2 with the GFP tag and to replace the galK insert in dbest1 with the GFP tag and the HA tag. 
 
 
 

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