400 PRODUCTION OF TRANSGENIC CLONED DOMESTIC CAT EMBRYOS BY LENTIVECTOR-MEDIATING TRANSGENESIS

2007 ◽  
Vol 19 (1) ◽  
pp. 316 ◽  
Author(s):  
M. C. Gómez ◽  
R. Kutner ◽  
D. Ricks ◽  
C. E. Pope ◽  
C. Dumas ◽  
...  
Keyword(s):  
Mammalian Species ◽  
Green Fluorescence Protein ◽  
Fluorescein Isothiocyanate ◽  
Domestic Cat ◽  
Epifluorescence Microscopy ◽  
Egfp Expression ◽  
Green Fluorescence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Fetal Fibroblasts

The domestic cat is a useful biomedical model because several cat diseases are analogous to inherited human disorders. Transgenic embryos have been produced by microinjecting lentivirus vectors (LV) carrying specific genes into the perivitelline space of mature oocytes or zygotes of different mammalian species (Hofmann et al. 2003 EMBO Rep. 4, 1054). One drawback of this approach is that integration of the transgene may not occur in each blastomere, and that mosaic embryos are formed (Kubish et al. 2006 Reprod. Fertil. Dev. 18, 295). Donor nuclei derived from cells stably transduced with a LV may provide a more effective strategy for producing transgenic animals via nuclear transfer (NT). The purpose of the present study was to determine the uselfulness of LV to deliver transgene into cat fetal fibroblasts (CFF) and to produce transgenic domestic cat cloned embryos expressing enhanced green fluorescence protein (eGFP). CFF were transduced with LV carrying the eGFP transgene. The LV-construct contained either the human cytomegalovirus (CMV) or the human translation elongation factor 1 alpha (hEF1alpha) promoter to achieve ubiquitous expression of the eGFP transgene. CFF at passage 1–2 were transduced with either LV-CMV or LV-hEF1alpha for 24 h. Cells expressing eGFP were observed at 24 and 48 h after co-incubation with the LV. Stable transgene expression in transduced CFF was observed and only those CFF that fluoresced green by epifluorescence microscopy with a fluorescein isothiocyanate (FITC) filter were selected for NT. Cleavage rate and embryo development to blastocyst stage (Day 8), respectively, of embryos reconstructed with transduced CFF from LV-CMV (79%; 12%) and LV-hEF1alpha (77%; 29%) were not different from those of cloned embryos reconstructed with non-transduced CFF cells (81%; 19%). None of the LV-CMV-derived cloned embryos expressed detectable levels of eGFP, whereas 18% of the LV-hEF1alpha-cloned embryos expressed detectable eGFP. Fluorescence in cloned embryos reconstructed with LV-hEFP1alpha promoter was not observed during the first 24–36 h, but from Day 2, three embryos (9%) at the 2-cell stage started to express eGFP. Two embryos fluoresced brightly and retained fluorescence through development to the morula stage at Day 7. The third embryo had faint levels of fluorescence until Day 5. On day 5, three other embryos (9%) showed faint fluorescence that disappeared by Day 7. Blastocysts at Day 8 derived from either construct did not exhibit green fluorescence. To analyze lentiviral integration and the number of proviral integrants, real-time PCR quantification was performed on genomic DNA of single blastocysts. The number of provirus copies present in the genome of LV-CMV-(n = 4) and LV-hEF1alpha-(n = 6) derived cloned blastocysts ranged from 5 to 9 and 3 to 9 copies, respectively, whereas cloned blastocysts (n = 2) using non-transduced CFF were negative. In summary, we have established that transgenic domestic cat cloned embryos can be produced. All cloned blastocysts derived from either LV construct carried the provirus. However, eGFP expression was not observed in the blastocysts, possibly due to transgene silencing.

scholarly journals 58 HANDMADE CLONING IN TRANS-SPECIES NT: CULTURE MEDIUM HAS AN EFFECT ON THE ABILITY OF RECONSTRUCTED BOVINE-MURINE EMBRYOS TO DEVELOP BEYOND THE 8-CELL STAGE

2005 ◽  
Vol 17 (2) ◽  
pp. 179 ◽  
Author(s):  
P.-M. Nieminen ◽  
M. Aho ◽  
K. Kananen-Anttila ◽  
E. Reinikainen ◽  
M. Halmekytö
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Reconstructed Embryos ◽  
Fetal Fibroblasts ◽  
Morula Stage ◽  
In Trans ◽  
Murine Embryos ◽  
Species Specific

The objectives of studies of trans-species nuclear transfer (NT) include epigenetic reprogramming and stem cell technology. The present study evaluated the effect of culture media on the development of reconstructed bovine-murine embryos. Bovine NT served as a technical control. The NT embryos were produced with the hand made cloning (HMC) technique (Vajta G et al. 2003 Biol. Reprod. 68, 571–578), and to our knowledge, this is the first report on the application of HMC in trans-species NT. Abattoir-derived bovine oocytes were matured for 21 h and enucleated by hand as described (Vajta G et al. 2003). Bovine cytoplasts were fused with either bovine granulosa cells or murine fetal fibroblasts. The bovine NT embryos were cultured for 7 days in modified SOFaaci (Holm P et al. 1999 Theriogenology 52, 683–700) containing either 5% FBS (8 trials) or 4 mg mL−1 fatty acid free albumin (FAFBSA, 6 trials). Bovine-murine NT embryos were cultured for 4.5 days in SOFaaci + FAFBSA (5 trials), or for the first 12 h in SOFaaci + FAFBSA and until 4.5 days in KSOMaa (Biggers JD 1991 J. Reprod. Fertil. 91, 543) containing 1 mg mL−1 embryo-tested BSA (4 trials). The results are shown in Table 1. In bovine NT embryos, both cleavage (day 2) and day 7 blastocyst rates were significantly improved was SOFaaci + FAFBSA was used as culture medium. Culture medium did not affect the cleavage rate of bovine-murine NT embryos at 12–16 h after start of culture. The development of reconstructed bovine-murine embryos beyond the 8-cell stage was significantly improved when SOFaaci + FAFBSA was replaced with KSOMaa + BSA after 12 h culture. Fourteen of a total of 464 (3.0%) Day 4.5 bovine-murine reconstructed embryos reached early morula stage with signs of compaction. The study showed that the development of the reconstructed NT embryos was significantly affected by the culture medium. Contrary to earlier findings (Park SH et al. 2004 Mol. Reprod. Dev. 68, 25–34), the bovine-murine reconstructed embryos developed beyond 8-cell stage, even until early compaction. The gene expression of species-specific and development-related genes of the reconstructed embryos is under characterization. Table 1. Effect of culture medium on cleavage and development of reconstructed NTt embryos Professor Gabor Vajta is greatly acknowledged for his contribution in establishing the HMC technique in our laboratory.

402 OOCYTE-MEDIATED GENE TRANSFER: A NOVEL APPROACH TO PRODUCE TRANSGENIC PORCINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 317
Author(s):  
M. K. Gupta ◽  
S. J. Uhm ◽  
E. Y. Kim ◽  
Y. H. Jung ◽  
J. Y. Yu ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Fluorescent Protein ◽  
Standard Procedure ◽  
Metaphase Plate ◽  
Egfp Expression ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Transgenic Livestock ◽  
Expression Rate

Classical approaches for producing transgenic livestock require labor-intensive, time-consuming, and expensive methods but have low transgenic efficiency and a high mosaicism rate. This study evaluated a simplified method for producing transgenic porcine embryos by microinjecting a DNA construct into unfertilized metaphase oocytes that were subsequently fertilized in vitro. For this, oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro for 42–44 h and were microinjected with DNA solution (10 ng �L-1) using a femtojet microinjector (Eppendorf, Hamburg, Germany). The DNA (4.7 kb) was derived from the pEGFP-C1 plasmid (Clontech Laboratories Inc., Palo Alto, CA, USA), which contains the enhanced green fluorescent protein (EGFP) encoding transgene under the control of cytomegalovirus promoter, and linearized with ApaLI restriction enzyme. Injected oocytes were then in vitro-fertilized using fresh epididymal sperm obtained from abattoir-derived porcine testis by standard procedure and cultured in NSCU23 medium supplemented with 0.4% BSA. The efficiency of transgenesis was monitored by visualization of green florescence under UV illumination using a EGFP filter set. Data were analyzed by Student's t-test. Results showed that the cleavage rate of injected oocytes (68.7 ± 0.5%) was similar to that of non-injected control oocytes (67.8 ± 0.4%). However, a high percentage of injected oocytes showed a developmental block at the 2–4 cell stage. The EGFP expression rate at 2–4 cell stage, when expressed as proportion of injected oocyte, was 17.2 ± 0.1%. Interestingly, mosaicism was not observed. The EGFP expression rate increased to 26.7 ± 0.1% when the DNA concentration was increased to 40 ng µL−1. Injecting the DNA solution near the metaphase plate of the oocyte did not improve (P < 0.05) the EGFP expression rate (22.2 ± 0.1%). A high proportion of EGFP-expressing oocytes blocked at the 4–8 cell stage and did not progress to blastocyst, possibly due to random integration of the transgene in developmentally important gene loci. Our results thus suggest oocyte-mediated gene transfer as a promising tool for producing transgenic livestock. However, further research is required to improve its efficiency. This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.

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.

scholarly journals Signalling pathways and mechanistic cues highlighted by transcriptomic analysis of primordial, primary, and secondary ovarian follicles in domestic cat

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shauna Kehoe ◽  
Katarina Jewgenow ◽  
Paul R. Johnston ◽  
Susan Mbedi ◽  
Beate C. Braun
Keyword(s):  
Gene Expression ◽  
Transforming Growth Factor ◽  
Ovarian Follicle ◽  
Mammalian Species ◽  
Expression Patterns ◽  
Ovarian Follicles ◽  
Domestic Cat ◽  
Transcriptional Analysis ◽  
Ovarian Folliculogenesis

AbstractIn vitro growth (IVG) of dormant primordial ovarian follicles aims to produce mature competent oocytes for assisted reproduction. Success is dependent on optimal in vitro conditions complemented with an understanding of oocyte and ovarian follicle development in vivo. Complete IVG has not been achieved in any other mammalian species besides mice. Furthermore, ovarian folliculogenesis remains sparsely understood overall. Here, gene expression patterns were characterised by RNA-sequencing in primordial (PrF), primary (PF), and secondary (SF) ovarian follicles from Felis catus (domestic cat) ovaries. Two major transitions were investigated: PrF-PF and PF-SF. Transcriptional analysis revealed a higher proportion in gene expression changes during the PrF-PF transition. Key influencing factors during this transition included the interaction between the extracellular matrix (ECM) and matrix metalloproteinase (MMPs) along with nuclear components such as, histone HIST1H1T (H1.6). Conserved signalling factors and expression patterns previously described during mammalian ovarian folliculogenesis were observed. Species-specific features during domestic cat ovarian folliculogenesis were also found. The signalling pathway terms “PI3K-Akt”, “transforming growth factor-β receptor”, “ErbB”, and “HIF-1” from the functional annotation analysis were studied. Some results highlighted mechanistic cues potentially involved in PrF development in the domestic cat. Overall, this study provides an insight into regulatory factors and pathways during preantral ovarian folliculogenesis in domestic cat.

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 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.

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.

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