The role of post-transcriptional RNA processing and plasmid vector sequences on transient transgene expression in zebrafish.
A tissue-specific transgenic model was employed to test the effects of intron and vector sequences on transgene expression in zebrafish after microinjection. In this model, the 2.3 kb promoter taken from the 5' upstream region of the transcription initiation site of keratin 4 (krt4) was used to drive the enhanced green fluorescence protein (EGFP) reporter gene in a transgenic vector. For assaying the strength of EGFP expression, the effects of including an intron before the EGFP coding region or using different forms of DNA, including circular plasmid, linear full-length plasmid, and the linear transgene coding region without any prokaryotic vector sequence, were tested. After microinjection, the transgene expression was analyzed using transient assays. Consequently, further comparative analysis supported by Fisher's exact test was performed based on the data generated by analyzing the strength of the transgene expression. It was shown that inclusion of an intron in the construct increases the transgene expression in a transient transgenic zebrafish assay. Furthermore, the circular plasmid containing the transgene produced the strongest EGFP expression.
Chatterjee
Sumantra
S
Stem Cell and Developmental Biology, Genome Institute of Singapore, 60 Biopolis Street, Singapore, 138672, Singapore.
Min
Lin
L
Karuturi
R Krishna Murthy
RK
Lufkin
Thomas
T
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007438:Introns / Q000235:genetics; D053527:Keratin-4 / Q000235:genetics / Q000378:metabolism; D008845:Microinjections; D010957:Plasmids / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional; D011817:Rabbits; D019076:Transgenes / Q000235:genetics / Q000502:physiology*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D055544:beta-Globins / Q000235:genetics
D053527:Keratin-4; D055544:beta-Globins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins