Optimized transfection of mRNA transcribed from a d(A/T)100 tail-containing vector.
Studies employing mRNA transfection are currently limited by a lack of transcription vectors for generating a long poly(A) tail-containing mRNA and published methods for efficient mRNA transfection. We have constructed a transcription vector containing firefly luciferase gene (pBS-FLuc-A100) to generate luciferase mRNA with A100 tail followed by no heterologous sequence. The pBS-FLuc-A100 was propagated in XL1-Blue, in which the plasmid was more stable than in other bacterial strains. Optimal mRNA transfection conditions were determined using TransMessenger Transfection Reagent (Qiagen) and yeast tRNA as a carrier. Firefly luciferase expression, which peaked at about 12 h post-transfection, was detected with as little as 5 ng mRNA and was linear with mRNA amount up to 100 ng. When cells were transfected with luciferase mRNA containing different lengths of poly(A) tail, luciferase expression increased proportionally with poly(A) tail length up to 60A residues and then declined. Cell lines from monkey, mouse, and rat were transfected efficiently by this method. Like cellular ferritin heavy chain mRNA, which contains an iron response element in its 5'UTR, translation of transfected luciferase mRNA containing the 5'UTR of ferritin mRNA was iron-dependent. Our results demonstrate that the poly(A) vector and the transcription method described will be useful to study the regulation of gene expression at the mRNA level by UTRs.
Elango
Narayanasamy
N
Geriatric Research Education and Clinical Center, South Texas Veterans Health Care System, Audie L. Murphy Division, Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA. elangon@uthscsa.edu
Elango
Suratha
S
Shivshankar
Pooja
P
Katz
Michael S
MS
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D009928:Organ Specificity; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification; D011061:Poly A / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics*; D051381:Rats; D013997:Time Factors; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection / Q000295:instrumentation / Q000379:methods*
D012333:RNA, Messenger; D011061:Poly A; D004247:DNA; D008156:Luciferases