326 GENERATION OF A DOUBLE-TRANSGENIC PIG WITH PANCREAS-SPECIFIC GREEN AND LIVER-SPECIFIC RED FLUORESCENCE

Transgenic (Tg) pigs with organ/tissue-specific fluorescence expression provide invaluable research tools for many types of studies, such as organogenesis analysis, in vitro tissue generation from pluripotent cells, and progenitor/stem cell transplantation therapy. We aimed to develop a Tg pig characterised by pancreas- and liver-specific fluorescence expression. A 8.4 kb transgene construct expressing Venus (green fluorescence) under the control of the mouse Pdx1 (pancreatic duodenal homeobox-1) promoter and a BAC-derived construct (170 kb) consisting of the whole-length porcine albumin (Alb) promoter and humanized Kusabira-Orange (huKO, red fluorescence) was introduced into porcine in vitro-matured oocytes using the intracytoplasmic sperm injection (ICSI)-mediated gene transfer method. Injected embryos were transferred to the oviducts of oestrus-synchronized recipients after culture for 1 to 3 days. The transfer of 370 Pdx1-Venus embryos into 4 recipients produce 22 (5.9%) fetuses/piglets, and 9 (40.9%) Tg pigs exhibited pancreas-specific Venus expression. Two (1 male and 1 female) founder Pdx1-Venus-Tg pigs were mated with wild-type (WT) pigs and produced 32 offspring in 3 litters, of which 16 (50.0%) were transgenic. Pancreas-specific Venus expression was inherited in these Tg offspring. The transfer of 523 Alb-huKO embryos into 4 recipients resulted in 19 (3.6%) piglets including a Tg female, which showed liver-specific huKO fluorescent expression. Expression of huKO was detected by RT-PCR exclusively in liver, but not in 7 other organs/tissues examined, including heart, lung, stomach, small intestine, spleen, kidney and skin. This founder Tg female produced a total of 12 non-Tg and 5 Tg offspring (in 2 litters) after mating with a WT boar. Liver-specific huKO expression was inherited in these Tg offspring. Furthermore, the mating of a female Pdx1-Venus pig with an Alb-huKO boar yielded 7 non-Tg and 10 Tg pigs. Four of these Tg pigs carrying both of the transgenes exhibited both pancreas-specific Venus and liver-specific huKO expression in single individuals. Double-Tg pigs with pancreas-specific green fluorescence and liver-specific red fluorescence grew normally, and tests of their reproduction ability are currently underway. These data demonstrate that transgene introduction by ICSI-mediated gene transfer into in vitro-matured oocytes is a feasible option for generating pigs expressing fluorescent proteins in a tissue-specific manner.

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Transfection of Human Keratinocytes In Vitro with the Particle Mediated Gene Transfer and the Liposome-Mediated Gene Transfer Method

Biological Matrices and Tissue Reconstruction ◽

10.1007/978-3-642-60309-9_32 ◽

1988 ◽

pp. 267-278

Author(s):

Ch. Andree ◽

E. Tanczos ◽

B. Flis ◽

D. Schaefer ◽

J. Kopp ◽

...

Keyword(s):

Gene Transfer ◽

Human Keratinocytes ◽

Transfer Method ◽

Gene Transfer Method

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Factors affecting the efficiency of producing porcine embryos expressing enhanced green fluorescence protein by ICSI-mediated gene transfer method

Animal Reproduction Science ◽

10.1016/j.anireprosci.2008.08.014 ◽

2009 ◽

Vol 113 (1-4) ◽

pp. 156-166 ◽

Cited By ~ 12

Author(s):

Yi Wu ◽

Cheng-Jun Liu ◽

Peng-Cheng Wan ◽

Ze-Dong Hao ◽

Shen-Ming Zeng

Keyword(s):

Gene Transfer ◽

Green Fluorescence Protein ◽

Green Fluorescence ◽

Enhanced Green Fluorescence Protein ◽

Factors Affecting ◽

Fluorescence Protein ◽

Transfer Method ◽

Gene Transfer Method

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Organ/tissue-specific changes in the mitochondrial genome organization of in-vitro cultures derived from different explants of a single wheat variety

Theoretical and Applied Genetics ◽

10.1007/bf00223837 ◽

1992 ◽

Vol 85 (1) ◽

pp. 1-8 ◽

Cited By ~ 10

Author(s):

M. C. Morère-Le Paven ◽

Y. Henry ◽

J. De Buyser ◽

F. Corre ◽

C. Hartmann ◽

...

Keyword(s):

Mitochondrial Genome ◽

Genome Organization ◽

Wheat Variety ◽

In Vitro Cultures ◽

Tissue Specific ◽

Organ Tissue

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Novel in vitro gene transfer method for study of local modulators in vascular smooth muscle cells.

Hypertension ◽

10.1161/01.hyp.21.6.894 ◽

1993 ◽

Vol 21 (6_pt_2) ◽

pp. 894-899 ◽

Cited By ~ 35

Author(s):

R Morishita ◽

G H Gibbons ◽

Y Kaneda ◽

T Ogihara ◽

V J Dzau

Keyword(s):

Smooth Muscle ◽

Gene Transfer ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells ◽

Transfer Method ◽

Gene Transfer Method

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Two-color flow-cytometric analysis of the growth cycle of Plasmodium falciparum in vitro: identification of cell cycle compartments.

Journal of Histochemistry & Cytochemistry ◽

10.1177/34.12.2431031 ◽

1986 ◽

Vol 34 (12) ◽

pp. 1651-1658 ◽

Cited By ~ 19

Author(s):

J D Hare

Keyword(s):

Cell Cycle ◽

Plasmodium Falciparum ◽

Dna Synthesis ◽

Flow Cytometric Analysis ◽

Growth Cycle ◽

Green Fluorescence ◽

Red Fluorescence ◽

Color Display ◽

Flow Cytometric

A previous study (Hare JD, Bahler DW: J Histochem Cytochem 34:215, 1986) has shown that the flow cytometric analysis of acridine-orange-stained Plasmodium falciparum growing in vitro generates a complex two-color display, regions of which correlate with the major morphological stages. In this report, four cell cycle compartments (A-D) are defined by characteristic ratios of red and green fluorescence of cells distributed throughout the erythrocytic cycle as well as by the differential effects of several metabolic inhibitors. The primary characteristic of cells in compartment A is the significant increase in red fluorescence. Inhibition of DNA synthesis by either aphidicolin or hydroxyurea causes the accumulation of cells at the interface between compartments A and B, whereas n-butyrate prevents cells in compartment A from reaching the A-B interface. Cells in compartment A display a small increase in green fluorescence which is independent of DNA synthesis but is enhanced by n-butyrate treatment. Cells in compartment B display a continued increase in red fluorescence coupled with a significant increase in green fluorescence, reflecting the onset of DNA synthesis in compartment B. The transition to compartment C is more abrupt and is associated with a marked increase in green fluorescence and little increase in red fluorescence. Compartment D is characterized by an increase in red fluorescence and a continued rise in green fluorescence. It is postulated that these discontinuities in the two-color display reflect not only changes in the rates of RNA and DNA synthesis but also decondensation of parasite chromatin in compartment A as the organism prepares for DNA synthesis, and re-condensation in compartment D as the newly replicated chromatin prepares for segregation into merozoites. The method described promises to provide a sensitive and rapid technique to study the effects of various factors on the growth cycle of the parasite.

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rAAV Vectors as Safe and Efficient Tools for the Stable Delivery of Genes to Primary Human Chondrosarcoma CellsIn VitroandIn Situ

Sarcoma ◽

10.1155/2012/347417 ◽

2012 ◽

Vol 2012 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Henning Madry ◽

Jagadeesh K. Venkatesan ◽

Gertrud Schmitt ◽

Sarah Schetting ◽

Myriam Ekici ◽

...

Keyword(s):

Gene Transfer ◽

Safe Delivery ◽

E Coli ◽

Human Chondrosarcoma ◽

Gene Vectors ◽

Transfer Strategies ◽

Transfer Method ◽

Metabolic Activities

Treatment of chondrosarcoma remains a major challenge in orthopaedic oncology. Gene transfer strategies based on recombinant adenoassociated viral (rAAV) vectors may provide powerful tools to develop new, efficient therapeutic options against these tumors. In the present study, we tested the hypothesis that rAAV is adapted for a stable and safe delivery of foreign sequences in human chondrosarcoma tissue by transducing primary human chondrosarcoma cellsin vitroandin situwith different reporter genes (E. coli lacZ, fireflyluc, Discosoma sp.RFP). The effects of rAAV administration upon cell survival and metabolic activities were also evaluated to monitor possibly detrimental effects of the gene transfer method. Remarkably, we provide evidence that efficient and prolonged expression of transgene sequences via rAAV can be safely achieved in all the systems investigated, demonstrating the potential of the approach of direct application of therapeutic gene vectors as a means to treat chondrosarcoma.

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Visualization ofToxoplasma gondiistage conversion by expression of stage-specific dual fluorescent proteins

Parasitology ◽

10.1017/s0031182009005836 ◽

2009 ◽

Vol 136 (6) ◽

pp. 579-588 ◽

Cited By ~ 15

Author(s):

A. UNNO ◽

K. SUZUKI ◽

T. BATANOVA ◽

S.-Y. CHA ◽

H.-K. JANG ◽

...

Keyword(s):

Fluorescent Protein ◽

Fluorescent Proteins ◽

Host Cells ◽

Live Cells ◽

Green Fluorescence ◽

Red Fluorescence ◽

Genes Encoding ◽

Transgenic Parasite ◽

P38 Mapk Inhibitor ◽

Green Fluorescent

SUMMARYTo recognize the stage conversion ofToxoplasma gondiibetween tachyzoite and bradyzoite in live host cells, a transgenicT. gondiiline, which expressed stage-specific red and green fluorescence, was constructed.T. gondiiPLK strain tachyzoites were stably transformed with genes encoding red fluorescent protein (DsRed Express) and green fluorescent protein (GFP) under the control of tachyzoite-specific SAG1 and bradyzoite-specific BAG1 promoters, respectively. The resulting transgenic parasite was designated PLK/DUAL. When PLK/DUAL was cultured in pH 7·0 medium, the PLK/DUAL zoites expressed red fluorescence, but no detectable levels of green fluorescence were observed. The PLK/DUAL zoites reacted with anti-SAG1 antibody, but not anti-BAG1 antiserum. When PLK/DUAL was cultured under high pH conditions, or in the presence of the p38 MAPK inhibitor SB202190, a small number of zoites expressed green fluorescence and were BAG1 positive. C57BL/6J mice were infected with PLK/DUAL tachyzoites. During the acute and reactivating phase, zoites expressed red fluorescence. However, green fluorescence was not detectable. By contrast, latent cysts expressed green fluorescence. The stage-specific dual fluorescence of PLK/DUAL facilitates identification of the parasitic stage in live cells, with the advantage that fixation or immunostaining is not required.

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