A Novel Method for the Production of Transgenic Cloned Pigs: Electroporation-Mediated Gene Transfer to Non-Cultured Cells and Subsequent Selection with Puromycin1

Abstract. For many years, ex vivo gene transfer has been used for genetic manipulation of various organs. In the kidney, ex vivo gene transfer was reported using mesangial cells and macrophages. In rats, cultured cells injected into the renal artery are accumulated selectively in the glomerulus. With this approach, it is possible to transfer genetically engineered cells to normal and diseased glomeruli. The transfer of genetically engineered cells to glomeruli can be used for several purposes. With the use of resident glomerular cells engineered in vitro, it is possible to examine how the cells that overexpress certain genes behave differently in normal and diseased glomeruli. Both gain-of-function and loss-of-function strategies are useful for this purpose. For the latter, stable expression of antisense cDNA, ribosomes, or dominant-negative mutants is available. By transfer of engineered cells producing secretory, recombinant proteins, it is possible to modify glomerular microenvironment in vivo. Transfer of genes encoding therapeutically relevant molecules could be useful for therapeutic intervention. Transfer of engineered leukocytes to the glomerulus also allows investigation of cross talk between leukocytes and resident cells. Transfer of stimulated leukocytes is useful for investigation of the pathologic actions of infiltrating cells on glomerular structure and function. Leukocytes in which certain gene functions are selectively reinforced or deleted would be useful for elucidation of the exact functions of leukocyte-associated genes in glomerular diseases. This article summarizes current experience with the adoptive transfer of engineered cells to the glomerulus for investigation of and therapy for glomerular diseases.

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Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells [published erratum appears in Blood 1995 Feb 1;85(3):862]

Blood ◽

10.1182/blood.v84.5.1492.bloodjournal8451492 ◽

1994 ◽

Vol 84 (5) ◽

pp. 1492-1500 ◽

Cited By ~ 1

Author(s):

S Goodman ◽

X Xiao ◽

RE Donahue ◽

A Moulton ◽

J Miller ◽

...

Keyword(s):

Gene Transfer ◽

Cultured Cells ◽

Vector System ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Hematopoietic Progenitors ◽

Coding Sequences ◽

Human Embryonic Kidney Cells ◽

Cell Genome

Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. We have now defined the ability of rAAV to introduce genes into primary hematopoietic progenitors. A vector was constructed containing the coding sequences for beta- galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong viral promotor. Infectious vector particles were prepared by cotransfection of the vector plasmid with a second plasmid that contained the coding sequences for AAV proteins into adenovirus-infected human embryonic kidney cells. These vector preparations transferred and expressed the beta-gal gene in human K562 erythroleukemia and Detroit 6 cells. Positive immunoselection yielded a population of enriched CD34+ cells that were transduced with the rAAV beta-gal vector. Nuclear localized enzyme expression was documented in 60% to 70% of infected cells. Progenitor-derived colonies that developed after 2 weeks in clonogenic cultures were shown to have viral- associated DNA at an estimated copy number of 1 to 2 per cell using a semiquantitative polymerase chain reaction (PCR) method. Integration of AAV into hematopoietic progenitors was documented using wild-type virus, as its genome may integrate at a preferred site on chromosome 19. Our data suggest that rAAV will transfer and express genes in primitive hematopoietic progenitors with high frequency, and support the development of this vector system for therapeutic gene transfer.

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Endothelial nitric oxide synthase gene transfer enhances dilation of newborn piglet pulmonary arteries

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1999.277.1.h371 ◽

1999 ◽

Vol 277 (1) ◽

pp. H371-H379

Author(s):

Judy L. Aschner ◽

Nora Kovacs ◽

James V. Perciaccante ◽

Jorge P. Figueroa ◽

Nishadi Thrikawala ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Gene Transfer ◽

Endothelial Nitric Oxide Synthase ◽

Cultured Cells ◽

Pulmonary Arteries ◽

Pulmonary Vasculature ◽

Enos Expression ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

We determined the expression and functional correlate of in vitro transfection with a recombinant adenoviral vector encoding the gene for bovine endothelial nitric oxide synthase (AdCMVeNOS) or Escherichia coliβ-galactosidase (AdCMVLacZ) in pulmonary endothelial cells (EC), vascular smooth muscle cells (VSMC), and pulmonary arteries (PA) from newborn piglets. AdCMVeNOS and AdCMVeLacZ vectors, grown in 293-cell monolayers, were purified by double-cesium gradient ultracentrifugation. Cell cultures and PA were incubated with increasing vector titers for 30 or 60 min, followed by incubation in fresh medium for 18 h at 37°C. LacZ expression was assessed by histochemical staining; eNOS expression was evaluated by Western blot analysis. Functional eNOS expression was determined by measurement of cGMP and quantification of the relaxation response to bradykinin (BK). In PA, LacZ transgene expression was preferentially localized to the adventitia and endothelium. Increased eNOS protein expression was observed in EC and VSMC transfected with AdCMVeNOS. Functional studies revealed increased cGMP abundance in cultured cells and enhanced relaxation to BK in AdCMVeNOS-transfected PA. These studies demonstrate that gene transfer with AdCMVeNOS results in functional expression and altered vasoactive responses in the neonatal pulmonary vasculature. Gene transfer with replication-deficient adenovirus vectors is a useful tool for the study of targeted genes in vascular biology.

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Visualization of Stat3 and Stat5 transactivation activity with specific response element dependent reporter constructs integrated into lentiviral gene transfer vectors

Hormone Molecular Biology and Clinical Investigation ◽

10.1515/hmbci.2010.017 ◽

2010 ◽

Vol 1 (3) ◽

Cited By ~ 1

Author(s):

Katrin Gäbel ◽

Nadja Lydia Bednorz ◽

Petra Klemmt ◽

Vida Vafaizadeh ◽

Corina Borghouts ◽

...

Keyword(s):

Gene Transfer ◽

Cancer Cells ◽

Cultured Cells ◽

Late Pregnancy ◽

Cellular Transformation ◽

Cell Types ◽

Specific Response ◽

Transactivation Activity ◽

Transfer Vectors ◽

Different Cell Types

Abstract: Signal transducer and activator of transcription 3 and 5 (Stat3 and Stat5) play important roles in cell differentiation, proliferation, apoptosis and inflammation. They are transiently activated by ligand-receptor interactions in normal cells but are often found to be constitutively active in cancer cells. Analysis of their activation pattern is therefore important for the description of developmental processes and the understanding of cellular transformation.: To visualize Stat3 and Stat5 transactivation activity in different cell types, we designed novel reporter constructs. These constructs comprise Stat3 or Stat5 specific promoter elements and reporter genes encoding β-galactosidase or fluorescent proteins. These constructs were integrated into lentiviral gene transfer vectors facilitating efficient transduction of most cell types.: The lentiviral reporter constructs were used to infect different cell types and their inducibility by activated Stat3 or Stat5 was measured. The Stat3-mCherry reporter was active in transduced tumor cells, which exhibit high levels of phosphorylated Stat3 and it was inducible in HepG2 liver cells by interleukin-6 treatment. The Stat5-LacZ reporter was active in cultured cells upon hormone induction of Stat5 and in primary mammary epithelial cells transplanted into cleared fat pads of mice during late pregnancy.: These novel reporter constructs are valuable tools to investigate and to distinguish between Stat3 and Stat5 activity in primary cells and cancer cells. They will also be useful in the discovery of drugs targeting Stat3 or Stat5. They can also be employed to generate transgenic mice and track Stat activity during development.

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