Int J Biochem Mol Biol 2013;4(4):201-208
Original Article
Insert sequence length determines transfection efficiency and gene expression levels in
bicistronic mammalian expression vectors
Andrew J Payne, Bryan C Gerdes, Simon Kaja, Peter Koulen
Vision Research Center, Department of Ophthalmology, Department of Basic Medical Science, University of Missouri School of Medicine, 2411
Holmes St, Kansas City, MO 64108, USA
Received November 12, 2013; Accepted December 1, 2013; Epub December 15, 2013; Published December 30, 2013
Abstract: Bicistronic expression vectors have been widely used for co-expression studies since the initial discovery of the internal ribosome
entry site (IRES) about 25 years ago. IRES sequences allow the 5’ cap-independent initiation of translation of multiple genes on a single
messenger RNA strand. Using a commercially available mammalian expression vector containing an IRES sequence with a 3’ green
fluorescent protein fluorescent marker, we found that sequence length of the gene of interest expressed 5’ of the IRES site influences both
expression of the 3’ fluorescent marker and overall transfection efficiency of the vector construct. Furthermore, we generated a novel construct
expressing two distinct fluorescent markers and found that high expression of one gene can lower expression of the other. Observations from
this study indicate that caution is warranted in the design of experiments utilizing an IRES system with a short 5’ gene of interest sequence
(<300 bp), selection of single cells based on the expression profile of the 3’ optogenetic fluorescent marker, and assumptions made during
data analysis. (IJBMB1310003).
Keywords: Bicistronic expression vectors, internal ribosome entry site, IRES sequences, transfection efficiency
Address correspondence to: Dr. Peter Koulen, Vision Research Center, University of Missouri School of Medicine, 2411 Holmes St, Kansas
City, MO 64108, USA. Tel: 816-404-1834; Fax: 816-404-1825; E-mail: koulenp@umkc.edu
Original Article
Insert sequence length determines transfection efficiency and gene expression levels in
bicistronic mammalian expression vectors
Andrew J Payne, Bryan C Gerdes, Simon Kaja, Peter Koulen
Vision Research Center, Department of Ophthalmology, Department of Basic Medical Science, University of Missouri School of Medicine, 2411
Holmes St, Kansas City, MO 64108, USA
Received November 12, 2013; Accepted December 1, 2013; Epub December 15, 2013; Published December 30, 2013
Abstract: Bicistronic expression vectors have been widely used for co-expression studies since the initial discovery of the internal ribosome
entry site (IRES) about 25 years ago. IRES sequences allow the 5’ cap-independent initiation of translation of multiple genes on a single
messenger RNA strand. Using a commercially available mammalian expression vector containing an IRES sequence with a 3’ green
fluorescent protein fluorescent marker, we found that sequence length of the gene of interest expressed 5’ of the IRES site influences both
expression of the 3’ fluorescent marker and overall transfection efficiency of the vector construct. Furthermore, we generated a novel construct
expressing two distinct fluorescent markers and found that high expression of one gene can lower expression of the other. Observations from
this study indicate that caution is warranted in the design of experiments utilizing an IRES system with a short 5’ gene of interest sequence
(<300 bp), selection of single cells based on the expression profile of the 3’ optogenetic fluorescent marker, and assumptions made during
data analysis. (IJBMB1310003).
Keywords: Bicistronic expression vectors, internal ribosome entry site, IRES sequences, transfection efficiency
Address correspondence to: Dr. Peter Koulen, Vision Research Center, University of Missouri School of Medicine, 2411 Holmes St, Kansas
City, MO 64108, USA. Tel: 816-404-1834; Fax: 816-404-1825; E-mail: koulenp@umkc.edu
 |  |  |  |  |  |  |