Electroporation of BmOR Promoters in Manduca Sexta Antennae

Abstract

Electroporation is a technique that employs electrical pulses to open pores in a cells membrane to allow DNA to enter and be transcribed using the cells machinery. A Bombyx mori odorant receptor (BmOr) promoter will be introduced to the antennae of Manduca sexta pupa using this technique. Vectors containing a BmOr promoter and TagRFP were constructed using pUC19 as the base plasmid by sequential ligations of first the promoter and then TagRFP using common restriction sites. After ligation the vectors were checked by restriction digest to extract and check the size of the insert and by sequencing the insert and comparing to the expected sequence. Vectors BmOR46-TagRFP-pUC19, BmOR41-TagRFP-pUC19, BmOR27-TagRFP-pUC19 were successfully created and are usable in electroporation experiments as demonstrated by successful electroporation of explanted antennal tissue. Future directions include use of GFP instead of TagRFP as the reporter gene and addition of RNAi to the vector to affect development through loss-of-function of a protein for the purpose of identifying some of the roles specific proteins play in olfactory development. Electroporation will be performed in live stage 4 pupa antenna instead of explants to allow for development of the antenna after introduction of the vector.