scholarly journals Methylguanine methyltransferase–mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model

2003 ◽  
Vol 112 (10) ◽  
pp. 1581-1588 ◽  
Author(s):  
Tobias Neff ◽  
Peter A. Horn ◽  
Laura J. Peterson ◽  
Bobbie M. Thomasson ◽  
Jesse Thompson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Animal Model ◽  
Large Animal Model ◽  
Large Animal ◽  
In Vivo Selection ◽  
Methylguanine Methyltransferase

Sustained Stable Engraftment of MGMT Gene-Modified Long-Term Repopulating Cells in Primary and Secondary Canine Recipients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 202-202
Author(s):  
Brian C. Beard ◽  
Reeteka Sud ◽  
Sabine Gerull ◽  
Laura J. Peterson ◽  
Tobias Neff ◽  
...  
Keyword(s):  
Stem Cells ◽  
Lentiviral Vector ◽  
Large Animal Model ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Mgmt Gene ◽  
In Vivo Selection ◽  
Therapeutic Benefits

Abstract Chemo-protection of gene-modified cells with mutant forms of methylguanine methyltransferase (MGMT) has the potential to increase the efficacy of clinical hematopoietic stem cell gene therapy. The therapeutic benefits of MGMT gene-modified cells are due to two factors: in vivo selection following transplantation to increase the level of cells that carry MGMT and a therapeutic transgene for genetic diseases or inhibitory transgenes for acquired diseases such as AIDS andhematopoietic protection from chemotherapy agents such as BCNU and temozolomide (TMZ) during treatment of malignant diseases. In the allogeneic and autologous canine models we have demonstrated efficient engraftment, in vivo selection, and chemo-protection of MGMT gene-modified cells and increased gene-marking levels to >90%. Unequivocal proof that true ‘stem cells’ are the transduced population leading to long-term engraftment and gene-marking is elusive and the only reliable test is sustained engraftment in the large animal model (i.e. canine or non-human primates). To further test the durability and engraftment potential of MGMT gene-modified canine cells, we investigated three aspects:engraftment and repopulation in a primary recipient,durability during in vivo selection, andengraftment and repopulation in a secondary recipient. Briefly, two DLA-identical allogeneic transplants were carried out with CD34-selected cells using an 18-hour transduction protocol. Cells were transduced with a VSVG-pseudotyped lentiviral vector expressing only the mutant version of MGMT(P140K). Cells were infused into primary recipients (G340 and G403) after pre-transplant conditioning with 200 cGy total body irradiation (TBI). G340 and G403 received multiple rounds of in vivo selection comprising 4 and 3 treatments with O6-benzylguanine (O6BG)/TMZ and 1 and 2 treatments with O6BG/BCNU respectively. Gene-marking stabilized in G340 and G403 after the final round of in vivo selection at about 60% and 4% respectively for at least 3 months before we proceeded to secondary transplantation. The original donors (G346 and G404) were conditioned with 920cGy TBI before receiving whole bone marrow cells from the primary recipients G340 and G403 respectively. Both G346 and G404 recovered neutrophil and platelet counts within expected time frames compared to historical controls. Shortly after transplantation the gene-marking in G346 stabilized around 80% and this level has been maintained for more than 1 year without in vivo selection. The gene-marking in G404 stabilized around 1% shortly after transplantation and this level was maintained until recently when in vivo selection was carried out in both G404 and G403 to increase gene-marking levels. Multiple clones contributed to hematopoietic repopulation in the primary and secondary recipients and we are in the process of tracking shared clones in the animals. These data establish the durability of MGMT(P140K) gene-modified cells and also present convincing evidence that true hematopoietic stem cells were transduced during the 18-hour transduction. This study demonstrates that MGMT(P140K) gene-modified cells repopulate a primary recipient and subsequently survive multiple rounds of in vivo selection while maintaining engraftment and repopulation potential in a secondary recipient.

A novel in vivo large animal model of lumbar spinal joint degeneration

2018 ◽  
Vol 18 (10) ◽  
pp. 1896-1909 ◽  
Author(s):  
Tian Wang ◽  
Matthew H. Pelletier ◽  
Chris Christou ◽  
Rema Oliver ◽  
Ralph J. Mobbs ◽  
...  
Keyword(s):  
Animal Model ◽  
Large Animal Model ◽  
Large Animal ◽  
Joint Degeneration ◽  
Lumbar Spinal

1 GENERATION OF A STABLE TRANSGENIC SWINE MODEL FOR CELL TRACKING AND CHROMOSOME DYNAMIC STUDIES

2016 ◽  
Vol 28 (2) ◽  
pp. 130
Author(s):  
R. Sper ◽  
S. Simpson ◽  
X. Zhang ◽  
B. Collins ◽  
J. Piedrahita
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Animal Model ◽  
Regenerative Medicine ◽  
Cancer Biology ◽  
Large Animal Model ◽  
Large Animal ◽  
Fetal Fibroblasts ◽  
Transgene Transmission

Transgenic pigs are an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic, and physiological similarities with humans. The development of a transgenic murine model with a fusion of green fluorescent protein (GFP) to histone 2B protein (H2B, protein of nucleosome core) resulted in an easier and more convenient method for tracking cell migration and engraftment levels after transplantation as well as a way to better understand the complexity of molecular regulation within cell cycle/division, cancer biology, and chromosome dynamics. Up to now the development of a stable transgenic large animal model expressing H2B-GFP has not been described. Our objective was to develop the first transgenic porcine H2B-GFP model via CRISPR-CAS9 mediated recombination and somatic cell nuclear transfer (SCNT). Porcine fetal fibroblasts were cotransfected with CRISPR-CAS9 designed to target the 3′ untranslated region of ACTB locus and a targeting vector containing 1Kb homology arms to ACTB flanking an IRES-H2B-GFP transgene. Four days after transfection GFP cells were fluorescence activated cell sorted. Single cell colonies were generated and analysed by PCR, and heterozygous colonies were used as donor cells for SCNT. The custom designed CRISPR-CAS9 knockin system demonstrated a 2.4% knockin efficiency. From positive cells, 119 SCNT embryos were generated and transferred to a recipient gilt resulting in three positive founder boars (P1 generation). Boars show normal fertility (pregnancies obtained via AI of wild type sows). Generated P1 clones were viable and fertile with a transgene transmission rate of 55.8% (in concordance with Mendel’s law upon chi-square test with P = 0.05). Intranuclear H2B-GFP expression was confirmed via fluorescence microscopy on 8-day in vitro cultured SCNT blastocysts and a variety of tissues (heart, kidney, brain, bladder, skeletal muscle, stomach, skin, and so on) and primary cultured cells (chondrocytes, bone marrow derived, adipocyte derived, neural stem cells, and so on) from P1 cloned boars and F1 42-day fetuses and viable piglets. In addition, chromosome segregation could be easily identified during cell cycle division in in vitro cultured stem cells. Custom designed CRISPR-CAS 9 are able to drive homologous recombination in the ACTB locus in porcine fetal fibroblasts, allowing the generation of the first described viable H2B-GFP porcine model via SCNT. Generated clones and F1 generation expressed H2B-GFP ubiquitously, and transgene transmission rates were with concordance of Mendel’s law. This novel large animal model represents an improved platform for regenerative medicine and chromosome dynamic and cancer biology studies.

scholarly journals Acute pressure changes in the brain are correlated with MR elastography stiffness measurements: initial feasibility in an in vivo large animal model

2017 ◽  
Vol 79 (2) ◽  
pp. 1043-1051 ◽  
Author(s):  
Arvin Arani ◽  
Hoon-Ki Min ◽  
Nikoo Fattahi ◽  
Nicholas M. Wetjen ◽  
Joshua D. Trzasko ◽  
...  
Keyword(s):  
Animal Model ◽  
Large Animal Model ◽  
Large Animal ◽  
Mr Elastography ◽  
Pressure Changes ◽  
The Brain
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