Expansion of EasyClone-MarkerFree toolkit for Saccharomyces cerevisiae genome with new integration sites

Abstract Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.

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Translational Regulation of Nuclear Gene COX4 Expression by Mitochondrial Content of Phosphatidylglycerol and Cardiolipin in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.26.3.743-753.2006 ◽

2006 ◽

Vol 26 (3) ◽

pp. 743-753 ◽

Cited By ~ 28

Author(s):

Xuefeng Su ◽

William Dowhan

Keyword(s):

Saccharomyces Cerevisiae ◽

Translational Regulation ◽

Nuclear Gene ◽

Green Fluorescence Protein ◽

Untranslated Regions ◽

Loss Of Function ◽

Green Fluorescence ◽

Recessive Mutations ◽

Protein Factor ◽

Fluorescence Protein

ABSTRACT Previous results indicated that translation of four mitochondrion-encoded genes and one nucleus-encoded gene (COX4) is repressed in mutants (pgs1Δ) of Saccharomyces cerevisiae lacking phosphatidylglycerol and cardiolipin. COX4 translation was studied here using a mitochondrially targeted green fluorescence protein (mtGFP) fused to the COX4 promoter and its 5′ and 3′ untranslated regions (UTRs). Lack of mtGFP expression independent of carbon source and strain background was established to be at the translational level. The translational defect was not due to deficiency of mitochondrial respiratory function but was rather caused directly by the lack of phosphatidylglycerol and cardiolipin in mitochondrial membranes. Reintroduction of a functional PGS1 gene under control of the ADH1 promoter restored phosphatidylglycerol synthesis and expression of mtGFP. Deletion analysis of the 5′ UTR COX4 revealed the presence of a 50-nucleotide fragment with two stem-loops as a cis-element inhibiting COX4 translation. Binding of a protein factor(s) specifically to this sequence was observed with cytoplasm from pgs1Δ but not PGS1 cells. Using HIS3 and lacZ as reporters, extragenic spontaneous recessive mutations that allowed expression of His3p and β-galactosidase were isolated, which appeared to be loss-of-function mutations, suggesting that the genes mutated may encode the trans factors that bind to the cis element in pgs1Δ cells.

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Determination of the topology of microsomal 17[beta]-hydroxysteroid dehydrogenase enzymes using redox-sensitive green fluorescence protein fusions

Endocrine Abstracts ◽

10.1530/endoabs.37.gp.05.03 ◽

2015 ◽

Author(s):

Maria Tsachaki ◽

Julia Birk ◽

Alex Odermatt

Keyword(s):

Green Fluorescence Protein ◽

Hydroxysteroid Dehydrogenase ◽

Green Fluorescence ◽

Fluorescence Protein

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Enhanced green fluorescence protein tracks trichosanthin in human choriocarcinoma cells as a feasible and stable reporter

Frontiers in Bioscience ◽

10.2741/1697 ◽

2005 ◽

Vol 10 (1-3) ◽

pp. 2279 ◽

Cited By ~ 4

Author(s):

Ye, Wang

Keyword(s):

Green Fluorescence Protein ◽

Green Fluorescence ◽

Enhanced Green Fluorescence Protein ◽

Fluorescence Protein ◽

Choriocarcinoma Cells ◽

Human Choriocarcinoma Cells

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Establishment of an Embryotoxicity Assay with Green Fluorescence Protein-expressing Embryonic Cell-derived Cardiomyocytes

Alternatives to Laboratory Animals ◽

10.1177/026119299902700303 ◽

1999 ◽

Vol 27 (3) ◽

pp. 471-484 ◽

Cited By ~ 3

Author(s):

Susanne Bremer ◽

Maaike Van Dooren ◽

Martin Paparella ◽

Eugen Kossolov ◽

Bernd Fleischmann ◽

...

Keyword(s):

Green Fluorescence Protein ◽

Embryonic Cell ◽

Green Fluorescence ◽

Fluorescence Protein

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Fertility of mice receiving vitrified adult mouse ovaries

Reproduction ◽

10.1530/rep.1.01030 ◽

2006 ◽

Vol 131 (4) ◽

pp. 681-687 ◽

Cited By ~ 13

Author(s):

Toshio Hani ◽

Takanori Tachibe ◽

Saburo Shingai ◽

Nobuo Kamada ◽

Otoya Ueda ◽

...

Keyword(s):

Germ Cells ◽

Adult Mouse ◽

Green Fluorescence Protein ◽

Green Fluorescence ◽

Experimental Animals ◽

Immature Mouse ◽

Estrous Cyclicity ◽

Fluorescence Protein ◽

High Degree ◽

Old Mice

Cryopreservation of the ovaries is a useful technology for preservation of germ cells from experimental animals, because if the female founder is infertile or has mutated mitochondrial DNA, preservation of female germ cells is necessary. Although it is possible to cryopreserve immature mouse ovaries with a high degree of viability by vitrification with a mixture of several cryoprotectants, the viability of cryopreserved adult mouse ovaries is still unknown. Here, we investigated the viability of mouse ovaries at various ages after cryopreservation by vitrification techniques. Donor ovaries were collected from 10-day-, 4-week-, 10-week- and 7-month-old, female, nulliparous, green fluorescence protein (GFP)-transgenic mice and cryopreserved by vitrification. The vitrified-warmed ovaries were orthotopically transplanted to 4- or 10-week-old mice. GFP-positive pups were obtained in all experimental groups. In the 4-week-old recipients, the percentages of GFP-positive pups among the total pups from recipients transplanted with ovaries of 10-day-, 4-week-, 10-week- and 7-month-old donors were 44%, 9%, 12% and 4% respectively. In the 10-week-old recipients, the percentages of GFP-positive pups among the total pups from recipients transplanted with ovaries of 10-day-, 4-week-, 10-week- and 7-month-old donors were 36%, 16%, 2% and 9% respectively. Furthermore, GFP-positive pups also were obtained from recipients transplanted with ovaries of donors without normal estrous cyclicity. Our results indicate that cryopreservation of mouse ovaries by vitrification is a useful method for the preservation of female germ cells from mice of various ages.

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Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/j.bbamcr.2015.04.002 ◽

2015 ◽

Vol 1853 (7) ◽

pp. 1672-1682 ◽

Cited By ~ 12

Author(s):

Maria Tsachaki ◽

Julia Birk ◽

Aurélie Egert ◽

Alex Odermatt

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Green Fluorescence Protein ◽

Endoplasmic Reticulum Membrane ◽

Green Fluorescence ◽

Fluorescence Protein

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Construction and Characterization of a Recombinant Human Respiratory Syncytial Virus Encoding Enhanced Green Fluorescence Protein for Antiviral Drug Screening Assay

BioMed Research International ◽

10.1155/2018/8431243 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Min Xu ◽

Yue-Ying Jiao ◽

Yuan-Hui Fu ◽

Nan Jiang ◽

Yuan-Bo Zheng ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Respiratory Tract ◽

Drug Screening ◽

Green Fluorescence Protein ◽

Respiratory Tract Disease ◽

Green Fluorescence ◽

Enhanced Green Fluorescence Protein ◽

Fluorescence Protein ◽

Syncytial Virus ◽

Tract Disease

Human respiratory syncytial virus (RSV) is the single most important cause of lower respiratory tract disease in infants and young children and a major viral agent responsible for respiratory tract disease in immunosuppressed individuals and the elderly, but no vaccines and antiviral drugs are available. Herein the recombinant RSV (rRSV) encoding enhanced green fluorescence protein (EGFP, rRSV-EGFP) was constructed and the potential for screening anti-RSV drugs was investigated. The recombinant plasmid of pBRATm-rRSV-EGFP, containing T7 transcription cassette composed of T7 promoter, RSV antigenomic cDNA with EGFP gene, HDV ribozyme (δ), and T7 terminator in the order of 5′ to 3′, was constructed and cotransfected into BHK/T7-9 cells together with helper plasmids encoding N, P, L, and M2-1 gene, respectively. The rescued rRSV-EGFP was confirmed by increasing expression of EGFP over blind passages and by RT-PCR. rRSV-EGFP was comparable to the other two recombinant RSVs encoding red fluorescent protein (RFP, rRSV-RFP) or luciferase (Luc, rRSV-Luc) in the growth kinetic, and there was a difference in sensitivity between them for screening anti-RSV agents based on infection of HEp-2 cells. The EGFP-encoding rRSV has been constructed and rescued successfully and has the potential for high-throughput anti-RSV drug screening in vitro.

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