Embryonic Stem Cell-Derived Pitx3-Enhanced Green Fluorescent Protein Midbrain Dopamine Neurons Survive Enrichment by Fluorescence-Activated Cell Sorting and Function in an Animal Model of Parkinson's Disease

Isolated blastomeres from 2- and 4-cell embryos are able to generate live offspring. However, the development of each cell of an 8-cell embryo is limited. Tetraploid embryos are used for aggregation with other embryos, embryonic stem cells, and iPS cells, and they are selected against during development of the fetal tissues, but persist in extraembryonic membranes. The objective of this work was to generate a new and simple method for cloning 8-cell bovine embryos and also to explore more efficient methods to multiply transgenic embryos by aggregation of each blastomere from a day-3 embryo with putative tetraploid embryos. To this aim, bovine cumulus–oocyte complexes were in vitro matured in standard conditions and subjected to IVF (day 0) according to Bracket and Oliphant (1975). After IVF, a group of presumptive zygotes was injected with ooplasmic vesicles incubated with 50 ng mL–1 of linearized pCX–egfp. Other group was cultured for 25 additional hours (day 1). At that time 2-cell embryos were electrofused twice at 40V for 25 μs at 100-ms intervals to generate putative tetraploid embryos, visualised as a single blastomere 1 h after the fusion pulse (fused embryos, F). Two aggregation groups were included. A synchronic group (S): IVF for the production of both transgenic embryos and fused embryos was done on the same day; and an asynchronic group (AS): IVF for transgenic embryos took place 1 day before IVF for fused embryos production, so embryos from the A group were younger. Controls consisted of the same S and AS groups, but no fusion was included (NF). On day 3, the enhanced green fluorescent protein [EGFP(+)] blastomeres were selected. Using the well of well system, 1 or 2 embryos of each fusion group (S or AS and F or NF) were removed of their ZP and aggregated in a microwell with one EGFP(+) blastomere from a 5- to 8-cell stage embryo (day 3). In vitro development of the aggregates and green fluorescent protein expression localization of blastocysts were analysed. Blastocysts were obtained for all groups; however, the 2A-F and 2A-NF groups showed the highest rates (44%, P < 0.05) compared with one embryo aggregation. The highest aggregation rates of the EGFP(+) blastomere were observed for 2A-F (67%) and 2A-NF (44%) groups, too. A very poor integration was noted in the 2S-NF (100%), 2S-F (94%), 1A-NF (89%), and 1S-NF (80%) groups. Localised EGFP distribution was also high in the 2A-F group (42%). In all cases, EGFP expression seemed to localise by the inner cell mass. We demonstrated that it is possible to multiply 8-cell embryos of genetic value and also transgenic embryos, in theory reducing mosaicism rates in future offspring. Moreover, our results give rise to the possibility of using EGFP like a reporter gene that could be used to evaluate aggregation efficiency by a fluorescence microscope.

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The Role of Lmx1a in the Differentiation of Human Embryonic Stem Cells into Midbrain Dopamine Neurons in Culture and After Transplantation into a Parkinson's Disease Model

Stem Cells ◽

10.1634/stemcells.2008-0734 ◽

2009 ◽

Vol 27 (1) ◽

pp. 220-229 ◽

Cited By ~ 55

Author(s):

Jingli Cai ◽

Angela Donaldson ◽

Ming Yang ◽

Michael S. German ◽

Grigori Enikolopov ◽

...

Keyword(s):

Parkinson’S Disease ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Disease Model ◽

Embryonic Stem ◽

Dopamine Neurons ◽

Midbrain Dopamine ◽

Midbrain Dopamine Neurons

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Essential Alterations of Heparan Sulfate During the Differentiation of Embryonic Stem Cells to Sox1-Enhanced Green Fluorescent Protein-Expressing Neural Progenitor Cells

Stem Cells ◽

10.1634/stemcells.2006-0445 ◽

2007 ◽

Vol 25 (8) ◽

pp. 1913-1923 ◽

Cited By ~ 82

Author(s):

Claire E. Johnson ◽

Brett E. Crawford ◽

Marios Stavridis ◽

Gerdy ten Dam ◽

Annie L. Wat ◽

...

Keyword(s):

Stem Cells ◽

Green Fluorescent Protein ◽

Embryonic Stem Cells ◽

Heparan Sulfate ◽

Progenitor Cells ◽

Neural Progenitor Cells ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Embryonic Stem ◽

Green Fluorescent

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Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors

Blood ◽

10.1182/blood-2008-04-149898 ◽

2009 ◽

Vol 113 (6) ◽

pp. 1241-1249 ◽

Cited By ~ 41

Author(s):

Enid Yi Ni Lam ◽

Jackie Y. M. Chau ◽

Maggie L. Kalev-Zylinska ◽

Timothy M. Fountaine ◽

R. Scott Mead ◽

...

Keyword(s):

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Hematopoietic Stem ◽

Blood Island ◽

Transgenic Lines ◽

Complex Regulation ◽

Green Fluorescent ◽

And Function ◽

Definitive Hematopoiesis ◽

Insight Into

Abstract The transcription factor Runx1 is essential for the development of definitive hematopoietic stem cells (HSCs) during vertebrate embryogenesis and is transcribed from 2 promoters, P1 and P2, generating 2 major Runx1 isoforms. We have created 2 stable runx1 promoter zebrafish-transgenic lines that provide insight into the roles of the P1 and P2 isoforms during the establishment of definitive hematopoiesis. The Tg(runx1P1:EGFP) line displays fluorescence in the posterior blood island, where definitive erythromyeloid progenitors develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the aorta-gonad-mesonephros, with enhanced green fluorescent protein–labeled cells later populating the pronephros and thymus. This suggests that a function of runx1 promoter switching is associated with the establishment of discrete definitive blood progenitor compartments. These runx1 promoter–transgenic lines are novel tools for the study of Runx1 regulation and function in normal and malignant hematopoiesis. The ability to visualize and isolate fluorescently labeled HSCs should contribute to further elucidating the complex regulation of HSC development.

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Tsc1-mTOR signaling controls the structure and function of midbrain dopamine neurons

10.1101/376814 ◽

2018 ◽

Author(s):

Polina Kosillo ◽

Natalie M. Doig ◽

Alexander H.C.W. Agopyan-Miu ◽

Kamran Ahmed ◽

Lisa Conyers ◽

...

Keyword(s):

Critical Role ◽

Dopamine Neurons ◽

Negative Regulator ◽

Gene Encoding ◽

Cognitive Behaviors ◽

High Prevalence ◽

Midbrain Dopamine ◽

And Function ◽

Mtor Complex 1 ◽

Midbrain Dopamine Neurons

SummarymTOR complex 1 (mTORC1) is a central coordinator of cell growth and metabolism. Mutations in regulators of mTORC1 cause syndromic disorders with a high prevalence of cognitive and psychiatric conditions. To elucidate the cellular origins of these manifestations, we conditionally deleted the gene encoding the mTORC1 negative regulator Tsc1 from mouse midbrain dopamine neurons, which modulate motor, affective, and cognitive behaviors that are frequently affected in psychiatric disorders. Loss of Tsc1 and constitutive activation of mTORC1 strongly impacted the properties of dopamine neurons, causing somatodendritic hypertrophy, reduced intrinsic excitability, altered axon terminal ultrastructure, and severely impaired dopamine release. These perturbations were associated with selective deficits in cognitive flexibility, which could be prevented by genetic reduction of the obligatory mTORC1 protein Raptor. Our results establish a critical role for mTORC1 in setting the functional properties of midbrain dopamine neurons, and indicate that dopaminergic dysfunction may underlie cognitive inflexibility in mTOR-related syndromes.

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144 Use of the CRISPR/CAS 9 system to produce porcine adipose-derived stem cells expressing enhanced green fluorescent protein

Reproduction Fertility and Development ◽

10.1071/rdv33n2ab144 ◽

2021 ◽

Vol 33 (2) ◽

pp. 180

Author(s):

Q. Xu ◽

D. J. Milner ◽

M. B. Wheeler

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Green Fluorescent Protein ◽

Cell Sorting ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Fluorescent Label ◽

Adipose Derived Stem Cells ◽

Egfp Gene ◽

Green Fluorescent

The goal of our project is to produce porcine adipose-derived stem cells (ASCs) stably expressing enhanced green fluorescent protein (eGFP) by using the clustered regularly interspaced short palindromic repeats (CRIPSR) technique. Fluorescent stem cells can facilitate the tracing and visualisation of stem cell migration, fusion, and participation in tissue regeneration after stem cell injection therapy, and represent a useful tool for tissue engineering research. The production of stem cells containing eGFP from ASCs using the CRISPR gene editing technique is able to reduce the time and labour requirement necessary for harvesting fluorescent cells from transgenic pigs. To generate fluorescent, edited cells, we utilised the ROSA 26 locus of pigs for insertion of the eGFP gene by homology-directed repair of Cas9-cleaved DNA at the ROSA 26 locus. The critical steps of producing stem cells expressing eGFP are (1) cloning of guide oligos into a Cas9 cutting vector and producing a repair template vector to insert GFP; (2) transfecting porcine stem cells with CRISPR plasmids; (3) cell sorting with flow cytometry to isolate colonies expressing GFP. A Rosa 26 Cas9-gRNA cutting vector was produced by cloning a guide RNA sequence into the vector backbone of plasmid pX458-GFP, and the donor vector was produced by the combination of the eGFP gene flanked with ROSA 26 genomic DNA inserted into plasmid pUC57. To isolate cells edited to contain the eGFP gene inserted into the ROSA-26 locus, we transfected 250,000 cells with a 1:1 mass mixture of Cas9-gRNA and eGFP repair plasmid using Lipofectamine STEM reagent (Invitrogen) in three trials. GFP+ cells were isolated by fluorescence-activated cell sorting, plated in 96-well plates, and monitored for colony growth and GFP expression. These trials produced an average of ∼70 colonies from sorting, and ∼1% GFP+ colonies. As pX458 drives expression of GFP as a marker for transfection, we hypothesised that we would potentially isolate more GFP+ edited colonies if we utilised a Cas9-gRNA cutting vector expressing mCherry and sorted for cells expressing both mCherry and GFP. This would allow enrichment of edited cells expressing GFP early after transfection, without interference of cells expressing GFP from the Cas9-gRNA vector alone. Utilising this method, we again obtained an average of ∼70 colonies from sorting, and 3% GFP+ colonies. Results were subjected to Student’s t-test. The comparisons were colonies/cell sorted and GFP+ colonies/cell sorted. All data were expressed as quadratic means+mean SE. When we compared groups, no differences were found for colonies/cell sorted: P=0.53 (1.11 E-03±9.16E-04 and 5.39 E-04±3.77 E-04, respectively, for green-green or red-green) and for GFP+ colonies/cell sorted: P=0.44 (1.94 E-05±2.15E-05 and 4.59 E-05±2.46 E-05, respectively, for green-green or red-green). In conclusion, our attempts to isolate ASC edited to express GFP have been successful, and our initial results suggest that utilising a dual fluorescent label sorting strategy does not enhance the number of GFP+ ASC colonies isolated. Future studies will verify that our GFP+ ASC retain normal stem cell properties.

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