338 BACTERIAL ARTIFICIAL CHROMOSOME (BAC) VECTORS FACILITATE EFFICIENT GENE TARGETTING IN KIDNEY CELLS OF PIG

2011 ◽  
Vol 23 (1) ◽  
pp. 264
Author(s):  
K. Wallner ◽  
A. Wuensch ◽  
K. Burkhardt ◽  
M. Kurome ◽  
B. Kessler ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Biomedical Research ◽  
Nuclear Transfer ◽  
Target Genes ◽  
Embryonic Stem ◽  
Artificial Chromosome ◽  
Site Directed Mutagenesis ◽  
Kidney Cells ◽  
Fetal Fibroblasts ◽  
Dmd Gene

Site-directed mutagenesis provided a powerful tool for studying gene functions in mice, but the lack of embryonic stem cells in other species limited the application of this technology to other species. Various attempts using negative selection, viral vectors, or other auxiliary means promoted specific projects but did not provide methods for routine experiments. Here, we describe a novel approach that enabled the site-directed modification of 3 different porcine genes relevant for biomedical research. Three main technologies were combined to achieve these goals: bacterial artificial chromosome (BAC) vectors, somatic cell transfection, and nuclear transfer (SCNT). BAC vectors contain large genomic regions in bacterial plasmids. They are superior to conventional targeting tools, as they provide extended regions of homology of several kilobases. Novel recombination tools using bacterial enzymes enable the modification of any DNA region of interest and thus allow the introduction of desired mutations into BACs. After verification of the wt-BAC sequence, it was altered by using modification vectors carrying the desired mutation. The modified BAC vectors are linearized and transfected after verification into primary kidney cell lines, and cells are selected for integration of the vectors. Kidney cells provide both good proliferation and high targeting rates, and thus improve the efficiency compared to fetal fibroblasts. Singularized clones are screened for the replacement of wild-type targeting loci by quantitative PCR. Targeted clones are used for SCNT and transfer of the resulting embryos into synchronized gilts. We have evaluated this technology by the modification of the porcine CFTR, GGTA1, and DMD genes. All 3 genes are relevant for biomedical research, as mutations in CFTR are causative for cystic fibrosis, the knockout of GGTA1 is essential for overcoming hyperacute rejection in xenotransplantation, and various deletions in the DMD gene are responsible for Duchenne muscular dystrophy. Gaining 13 targeted clones out of 1152 for CFTR, 9 out of 306 for GGTA1 and 6 out of 203 for DMD, we obtained efficiencies higher than 1% for each of the target genes. The power of our approach is underlined by the fact that CFTR and DMD are loci that are thought to be difficult to manipulate. The viability of targeted kidney cells and their suitability for nuclear transfer is accentuated by the pregnancy rates (2 out of 3) and the delivery of 4 to 10 piglets or fetuses in the case of CFTR and GGTA1. The heterozygous fetuses or piglets are verified by qPCR. In the case of the X-chromosomal DMD gene, we have generated the first full knockout by transfecting male cells. Pregnancies of a successfully targeted clone are under way. Thus, we consider the combination of modified BAC vectors, transfection of kidney cells, and nuclear transfer to be a technology with the potential for routine production of site-directed mutations. Supported by the Mukoviszidose e.V. and the Bayerische Forschungsstiftung.

scholarly journals 39 FACTORS INFLUENCING THE EFFICIENCY OF GENERATING GENETICALLY ENGINEERED PIGS BY NUCLEAR TRANSFER: MULTI-FACTORIAL ANALYSIS OF A LARGE DATA SET

2013 ◽  
Vol 25 (1) ◽  
pp. 167 ◽  
Author(s):  
M. Kurome ◽  
L. Geistlinger ◽  
B. Kessler ◽  
V. Zakhartchenko ◽  
N. Klymiuk ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Gene Targeting ◽  
Nuclear Transfer ◽  
Large Data ◽  
Genetically Engineered ◽  
Kidney Cells ◽  
Data Set ◽  
Large Data Set ◽  
Fetal Fibroblasts ◽  
Additive Gene

Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate genetically tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, including inducible transgene expression and gene targeting, the outcome in terms of live cloned piglets is quite variable. We used a large data set from 274 SCNT experiments (in total, 18 649 reconstructed embryos transferred into 193 recipients), performed over a period of 3 years, and evaluated it by multivariate analysis for combined assessment of multiple factors and their relative contribution to the efficiency of generation of genetically modified cloned pigs. Specifically, we addressed the effects of season, type of genetic modification (additive gene transfer v. gene targeting), donor cell source (mesenchymal stem cells, postnatal fibroblasts, fetal fibroblasts, and kidney cells), serial NT, and pre-selection of SCNT embryos for early development. Target parameters were pregnancy and delivery rates, as well as the numbers of born, live, and healthy offspring. Cloning efficiency was calculated as the number of cloned piglets relative to the number of transferred SCNT embryos. We used robust linear models adjusted to the underlying empirical distribution of the cloning outcome, as a straightforward approach to determine the statistically significant part of the network of factors affecting pig cloning. The SCNT experiments performed during the winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, whereas sequential NT and NT experiments performed during the summer decreased it. Although the effects of individual factors may be different between various laboratories, the statistical approach described in this study may help to identify and optimize the most critical specific factors to cloning success in programs aimed at the generation of genetically engineered pig models. Supported by the DFG (FOR535, FOR793), the Bayerische Forschungsstiftung, and Mukoviszidose e.V.

scholarly journals Targeted expression profiling reveals distinct stages of early canine fibroblast reprogramming are regulated by 2-oxoglutarate hydroxylases

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ian C. Tobias ◽  
Mian-Mian C. Kao ◽  
Thomas Parmentier ◽  
Hailey Hunter ◽  
Jonathan LaMarre ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Sendai Virus ◽  
Somatic Cells ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Experimental System ◽  
Cell Reprogramming ◽  
Limited Attention ◽  
Fetal Fibroblasts

Abstract Background Ectopic expression of a defined set of transcription factors allows the reprogramming of mammalian somatic cells to pluripotency. Despite continuous progress in primate and rodent reprogramming, limited attention has been paid to cell reprogramming in domestic and companion species. Previous studies attempting to reprogram canine cells have mostly assessed a small number of presumptive canine induced pluripotent stem cell (iPSC) lines for generic pluripotency attributes. However, why canine cell reprogramming remains extremely inefficient is poorly understood. Methods To better characterize the initial steps of pluripotency induction in canine somatic cells, we optimized an experimental system where canine fetal fibroblasts (cFFs) are transduced with the Yamanaka reprogramming factors by Sendai virus vectors. We use quantitative PCR arrays to measure the expression of 80 target genes at various stages of canine cell reprogramming. We ask how cFF reprogramming is influenced by small molecules affecting the epigenomic modification 5-hydroxymethylcytosine, specifically L-ascorbic acid and retinoic acid (AA/RA). Results We found that the expression and catalytic output of a class of 2-oxoglutarate-dependent (2-OG) hydroxylases, known as ten-eleven translocation (TET) enzymes, can be modulated in canine cells treated with AA/RA. We further show that AA/RA treatment induces TET1 expression and facilitates early canine reprogramming, evidenced by upregulation of epithelial and pluripotency markers. Using a chemical inhibitor of 2-OG hydroxylases, we demonstrate that 2-OG hydroxylase activity regulates the expression of a subset of genes involved in mesenchymal-to-epithelial transition (MET) and pluripotency in early canine reprogramming. We identify a set of transcription factors depleted in maturing reprogramming intermediates compared to pluripotent canine embryonic stem cells. Conclusions Our findings highlight 2-OG hydroxylases have evolutionarily conserved and divergent functions regulating the early reprogramming of canine somatic cells and show reprogramming conditions can be rationally optimized for the generation of maturing canine iPSC.

scholarly journals Targeted expression profiling reveals distinct stages of early canine fibroblast reprogramming are regulated by 2-oxoglutarate hydroxylases

2020 ◽  
Author(s):  
Ian C. Tobias ◽  
Mian-Mian C. Kao ◽  
Thomas Parmentier ◽  
Hailey Hunter ◽  
Jonathan LaMarre ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Sendai Virus ◽  
Somatic Cells ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Experimental System ◽  
Cell Reprogramming ◽  
Limited Attention ◽  
Fetal Fibroblasts

Abstract Background: Ectopic expression of a defined set of transcription factors allows the reprogramming of mammalian somatic cells to pluripotency. Despite continuous progress in primate and rodent reprogramming, limited attention has been paid to cell reprogramming in domestic and companion species. Previous studies attempting to reprogram canine cells have mostly assessed a small number of presumptive canine induced pluripotent stem cell (iPSC) lines for generic pluripotency attributes. However, why canine cell reprogramming remains extremely inefficient is poorly understood. Methods: To better characterize the initial steps of pluripotency induction in canine somatic cells, we optimized an experimental system where canine fetal fibroblasts (cFFs) are transduced with the Yamanaka reprogramming factors by Sendai virus vectors. We use quantitative PCR arrays to measure the expression of 80 target genes at various stages of canine cell reprogramming. We ask how cFF reprogramming is influenced by small molecules affecting the epigenomic modification 5-hydroxymethylcytosine, specifically L-ascorbic acid and retinoic acid (AA/RA). Results: We found that the expression and catalytic output of a class of 2-oxoglutarate-dependent (2-OG) hydroxylases, known as ten-eleven translocation (TET) enzymes, can be modulated in canine cells treated with AA/RA. We further show that AA/RA treatment induces TET1 expression and facilitates early canine reprogramming, evidenced by upregulation of epithelial and pluripotency markers. Using a chemical inhibitor of 2-OG hydroxylases, we demonstrate that 2-OG hydroxylase activity regulates the expression of a subset of genes involved in mesenchymal-to-epithelial transition (MET) and pluripotency in early canine reprogramming. We identify a set of transcription factors depleted in maturing reprogramming intermediates compared to pluripotent canine embryonic stem cells. Conclusions: Our findings highlight 2-OG hydroxylases have evolutionarily conserved and divergent functions regulating the early reprogramming of canine somatic cells and show reprogramming conditions can be rationally optimized for the generation of maturing canine iPSC.

Enriched Motor Neuron Populations Derived from Bacterial Artificial Chromosome-transgenic Human Embryonic Stem Cells

Neurosurgery ◽  
2008 ◽  
Vol 62 (6) ◽  
pp. 1400
Author(s):  
Dimitris G. Placantonakis ◽  
Mark Tomishima ◽  
Sabrina C. Desbordes ◽  
Fabien Lafaille ◽  
Nicholas D. Socci ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Bacterial Artificial Chromosome ◽  
Motor Neuron ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Artificial Chromosome ◽  
Neuron Populations

Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 67-72 ◽  
Author(s):  
So Gun Hong ◽  
Hyun Ju Oh ◽  
Jung Eun Park ◽  
Min Jung Kim ◽  
Geon A. Kim ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Transfected Cells ◽  
Fetal Fibroblasts ◽  
Transgenic Cells

SummarySomatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry ‘foreign’ DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8–16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8–16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine–bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.

scholarly journals Targeting CreERT2 expression to keratin 8-expressing murine simple epithelia using bacterial artificial chromosome transgenesis

2012 ◽  
Vol 21 (5) ◽  
pp. 1117-1123 ◽  
Author(s):  
Li Zhang ◽  
Boyu Zhang ◽  
Sang Jun Han ◽  
Amy N. Shore ◽  
Jeffrey M. Rosen ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Artificial Chromosome ◽  
Keratin 8

scholarly journals Revising the Chromosome-Specific Probes of White Hawk (Leucopternis albicollis)

Proceedings ◽  
2020 ◽  
Vol 76 (1) ◽  
pp. 1
Author(s):  
Ivanete Furo ◽  
Rafael Kretschmer ◽  
Jorge Pereira ◽  
Darren Griffin ◽  
Rebecca O’Connor ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Artificial Chromosome ◽  
Bird Of Prey ◽  
Inaccurate Data ◽  
Intrachromosomal Rearrangements

Leucopternis albicollis is a diurnal bird of prey with extensive karyotype reorganization. Chromosome-specific probes from this species have been used successfully to detect intrachromosomal rearrangements in different species of bird since 2010. However, some gaps were detected in this first set of probes. Here, we have obtained a new set of whole chromosome probes in order to improve the previous one; also, we have performed experiments using bacterial artificial chromosome (BAC) from chicken microchromosomes. Our results demonstrated that the microchromosomes were involved in fusion events. In addition, a new nomenclature has been proposed for the new set of probes and some inaccurate data were corrected.

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