Lack of excision of introns from primary transcripts of certain chicken vitellogenin II minigenes.
Two truncated versions of the chicken vitellogenin II gene VTGII were designed and constructed to include all known essential regulatory elements of the complete gene. Both pCB123 and pCB123/4 contain 945 base pairs (bp) of the 5'-flanking sequence, introns and exons 1-3, and a subset of the remaining 32 exons of VTGII, inserted into a pBluescript SK (+/-) vector. pCB123/4 contains 752 bp of legitimate VTGII 3'-flanking sequences, while the 3' end of pCB123 terminates at the VTGII cDNA end, followed by AT-tailing and vector sequences carried over during cloning. Expression of these plasmids was tested following their lipofection into primary cultures of chicken hepatocytes established from day 14 embryos. Poly(A)+ RNA derived from pCB123 was detected by Northern blotting and reverse transcription-polyacrylamide chain reaction. No evidence was observed for appropriate hormonal control of expression, despite the presence of 17 beta-estradiol or colipofection with the estrogen receptor clone pHEO. VTGII sequences at the 3' end of pCB123/4 led to an apparent destabilization of the RNA transcript. Unexpectedly, unprocessed pCB123 transcripts of varying lengths accumulated in the cells. These experiments constitute the first reported attempts to express authentic VTGII coding sequences in cultured cells and highlight the dilemma of which introns to include in a minigene. Despite reports that some minigenes are expressed more efficiently if one or two introns are included, other minigenes may be expressed more effectively in the absence of introns. In the case of a complex gene with many introns, such as VTGII, there may be a preferential order in which introns are removed from the primary construct. The truncation of complex genes to give functional minigenes for transgenic studies may require considerable experimentation.
Millan
C L
CL
Department of Animal and Poultry Science, University of Guelph, ON, Canada.
Gibbins
A M
AM
D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D002645:Chickens / Q000235:genetics*; D005091:Exons; D007438:Introns; D008099:Liver / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase; D012045:Regulatory Sequences, Nucleic Acid; D014162:Transfection; D014819:Vitellogenins / Q000235:genetics*
D012333:RNA, Messenger; D014819:Vitellogenins; D012194:RNA-Directed DNA Polymerase