scholarly journals Fertility of mice receiving vitrified adult mouse ovaries

Reproduction ◽  
2006 ◽  
Vol 131 (4) ◽  
pp. 681-687 ◽  
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.

Introduction and expression of foreign genes in cultured mouse embryonic gonads by electroporation

2002 ◽  
Vol 14 (5) ◽  
pp. 259 ◽  
Author(s):  
Yuri Nakamura ◽  
Miwako Yamamoto ◽  
Yasuhisa Matsui
Keyword(s):  
Germ Cells ◽  
Fluorescent Protein ◽  
Green Fluorescence Protein ◽  
Lacz Gene ◽  
Dependent Manner ◽  
Green Fluorescence ◽  
Gene Construct ◽  
Gfp Gene ◽  
Fluorescence Protein ◽  
Foreign Genes

To analyse the functions of genes that are expressed and potentially involved in the development of embryonic gonad cells, a method was developed by which foreign genes can be introduced and expressed in cultured mouse genital ridges. Genital ridges from mouse embryos at 12.5 days post coitus (dpc) were injected with plasmid DNA of a green fluorescence protein (GFP) gene construct and then placed between small electrodes. Rectangular pulses were charged to electroporate DNA into the cells. The treated genital ridges were cultured on a membrane of a culture insert, and GFP gene expression was observed under a fluorescence microscope. Green fluorescence protein expression in the genital ridges was found as early as 1 h after electroporation. Thereafter, the expression gradually increased, peaked after 1 day, and then decreased. A significant number of cells were, however, still positive for fluorescence even after 2 weeks in the culture, in which both gonads and germ cells had continued to develop. The GFP gene was expressed in 1–2% of cells in each genital ridge in a DNA concentration-dependent manner. In addition, we confirmed that an electroporated red fluorescent protein (DsRed) gene construct was expressed in GFP-expressing primordial germ cells in genital ridges of Oct-4/GFP transgenic embryos, although the DNA was mainly found in somatic cells in genital ridges. Finally, an expression vector containing the internal ribosome entry site–green fluorescent protein (IRES-GFP) gene was constructed. An inserted lacZ gene showed similar expression pattern to that of GFP. Using this vector, we can easily monitor the expression of an inserted gene of interest by GFP expression. Therefore, this experimental system could be useful for quick assays of gene function in genital ridges.

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

2021 ◽  
Author(s):  
Mahsa Babaei ◽  
Luisa Sartori ◽  
Alexey Karpukhin ◽  
Dmitrii Abashkin ◽  
Elena Matrosova ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Recombinant Dna ◽  
Green Fluorescence Protein ◽  
Cellular Growth ◽  
Green Fluorescence ◽  
Biotechnological Production ◽  
Yeast Saccharomyces Cerevisiae ◽  
Integration Sites ◽  
Fluorescence Protein ◽  
Integration Efficiency

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.

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

1999 ◽  
Vol 27 (3) ◽  
pp. 471-484 ◽  
Author(s):  
Susanne Bremer ◽  
Maaike Van Dooren ◽  
Martin Paparella ◽  
Eugen Kossolov ◽  
Bernd Fleischmann ◽  
...  
Keyword(s):  
Green Fluorescence Protein ◽  
Embryonic Cell ◽  
Green Fluorescence ◽  
Fluorescence Protein

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

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
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.

Microplate reader operating procedure v1

2021 ◽  
Author(s):  
Shuning Guo ◽  
Zhujun Wei
Keyword(s):  
Operating Procedure ◽  
Fluorescence Intensity ◽  
Green Fluorescence Protein ◽  
Green Fluorescence ◽  
Microplate Reader ◽  
Fluorescence Protein

Use Tecan Spark® multimode microplate reader to measure absorbance or fluorescence intensity of green fluorescence protein.

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