Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5

Beck, Ewald; Ludwig, Günther; Auerswald, Ennes A.; Reiss, Bernd; Schaller, Heinz

doi:10.1016/0378-1119(82)90023-3

Cited by 901 publications

(365 citation statements)

References 16 publications

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“…The 5.15-kb product of the inverse PCR, called T. vaginalis expression vector 1, was purified and digested with KpnI and BamHI. The coding regions of cat, luc, and neo were derived from pBLCAT3 (12), pDR100 (13), and pKm2 (14), respectively. Each plasmid was used as a template in PCR using primers with a KpnI (5Ј end primer) or a BamHI (3Ј end primer) restriction enzyme site added at their 5Ј ends.…”

Section: Methodsmentioning

confidence: 99%

Transient and selectable transformation of the parasitic protist Trichomonas vaginalis

Delgadillo

Liston

Niazi

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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We have developed methods to transiently and selectably transform the human-infective protist Trichomonas vaginalis. This parasite, a common cause of vaginitis worldwide, is one of the earlier branching eukaryotes studied to date. We have introduced three heterologous genes into T. vaginalis by electroporation and have used the 5 and 3 untranslated regions of the endogenous gene ␣-succinyl CoA synthetase B (␣-SCSB) to drive transcription of these genes. Transient expression of two reporter proteins, chloramphenicol acetyltransferase (CAT) or luciferase, was detected when electroporating in the presence of 50 g closedcircular construct. Optimal levels of expression were observed using Ϸ2.5 ؋ 10 8 T. vaginalis cells and 350 volts, 960 Fd for electroporation; however, other conditions also led to significant reporter gene expression. A time course following the expression of CAT in T. vaginalis transient transformants revealed the highest level of expression 8-21 hr postelectroporation and showed that CAT activity is undetectable using TLC by 99 hr postelectroporation. The system we established to obtain selectable transformants uses the neomycin phosphotransferase (neo) gene as the selectable marker. Cells electroporated with 20 g of the NEO construct were plated in the presence of 50 g͞ml paromomycin and incubated in an anaerobic chamber. The paromomycin-resistant colonies that formed within 3-5 days were cultivated in the presence of drug and DNA was isolated for analyses. The NEO construct was shown to be maintained episomally, as a closed-circle, at between 10-30 copies per cell. The ability to transiently and selectably transform T. vaginalis should greatly enhance research on this important human parasite.

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Section: Methodsmentioning

confidence: 99%

Transient and selectable transformation of the parasitic protist Trichomonas vaginalis

Delgadillo

Liston

Niazi

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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“…The /?-lactamase fragment spans the region encoding the mature protein except the C-terminal 39 amino acids, as determined by sequencing a Ba131 deletion (H. Hansen, Dissertation, University of Dfisseldorf 1990), between two EcoRI sites. The 5' EcoRI site is identical to that in pMEX (Hansen and Roggenkamp 1989) (Beck et al 1982) was inserted into the SalI site of pMEX, resulting in plasmid pMEK1. The region encoding the mature /%lactamase was replaced by the same/~-lactamase fragment that was used for constructing pDH237, but instead of the inserted EcoRI linker at the 3' end, it contains a dodecamer ClaI linker.…”

Section: Methodsmentioning

confidence: 99%

Targeting sequences of the two major peroxisomal proteins in the methylotrophic yeast Hansenula polymorpha

et al. 1992

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“…Human plasminogen activator cDNA (tPA) has also been expressed [31] from a dicistronic construct in CHO DUKX-Bll cells, and we have obtained expression of human clotting factor IX (FIX), E. coli chloramphenicol acetyltransferase (CAT) and mouse DHFR from di-and tricistronic vectors transfected into BHK cells (Berkner et al, in preparation). The neomycin gene (neo) from Tn5 [32] can be used as well to obtain stable transformants from polycistronic transcripts. Recently we used G418 selection together with both di-and tricistronic constructs encoding human neuropeptide Y (NPY) [33], PP and neo to obtain stable transformants in the neuroendocrine cell line AtT-20 (Wulff et al, in preparation).…”

Section: Discussionmentioning

confidence: 99%

Expression of human pancreatic polypeptide precursors from a dicistronic mRNA in mammalian cells

Boel¹,

Berkner²,

Nexø³

et al. 1987

FEBS Letters

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The ability of eukaryotic ribosomes to reinitiate translation at downstream AUG codons on polycistronic mRNAs was used to select transfected CHO clones that secreted a precursor to the human pancreatic polypeptide (PP). In the in vitro constructed transcription unit, a viral promoter directed the synthesis of a dicistronic mRNA. The PP cDNA was placed in the Y-part of this transcript, while a DHFR cDNA was placed 3' to the PP. This dicistronic expression unit was transfected into CHO cells, and methotrexate-resistant colonies were selected. RNA-blots verified that the PP precursor and DHFR were expressed from the same dicistronic mRNA. The CHO cells synthesized the hormone precursor and secreted it through the constitutive secretory pathway.

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Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5

Cited by 901 publications

References 16 publications

Transient and selectable transformation of the parasitic protist Trichomonas vaginalis

Transient and selectable transformation of the parasitic protist Trichomonas vaginalis

Targeting sequences of the two major peroxisomal proteins in the methylotrophic yeast Hansenula polymorpha

Expression of human pancreatic polypeptide precursors from a dicistronic mRNA in mammalian cells

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