Construction of a panel of chromosome-specific oligonucleotide probes (PRINS-primers) useful for the identification of individual human chromosomes in situ

1995 ◽  
Vol 71 (2) ◽  
pp. 142-147 ◽  
Author(s):  
J. Koch ◽  
J. Hindkjær ◽  
S. Kølvraa ◽  
L. Bolund
Keyword(s):  
Oligonucleotide Probes ◽  
Human Chromosomes ◽  
Individual Human

scholarly journals Selective transfer of individual human chromosomes to recipient cells.

1985 ◽  
Vol 5 (1) ◽  
pp. 140-146 ◽  
Author(s):  
P J Saxon ◽  
E S Srivatsan ◽  
G V Leipzig ◽  
J H Sameshima ◽  
E J Stanbridge
Keyword(s):  
Hypoxanthine Phosphoribosyl Transferase ◽  
Human Chromosomes ◽  
Isoenzyme Analysis ◽  
Phosphoribosyl Transferase ◽  
Microcell Hybrid ◽  
Multiple Copies ◽  
G 11 ◽  
Hybrid Clones ◽  
Individual Human

Two hypoxanthine phosphoribosyltransferase-deficient human cell lines, D98/AH-2 and HT1080-6TG, were stably transfected with pSV2 gpt, a plasmid containing the selectable marker Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco gpt). Hypoxanthine-aminopterin-thymidine-resistant transformants arose with a frequency of ca. 10(-6) and contained mostly single, but occasionally multiple, copies of the plasmid sequences. These transformants actively express the Eco gpt marker. Single chromosomes from two different HT1080 gpt transformants and one D98 gpt transformant, containing the integrated plasmid sequences, were transferred via microcell-mediated chromosome transfer to hypoxanthine phosphoribosyl transferase-deficient mouse A9 cells. The transferred human chromosomes were identified as 2, 4, and 22, by using a combination of G-11 staining, G-banding, isoenzyme analysis, and in situ hybridization. This system is being used to create a library of interspecies microcell hybrid clones, each clone containing a unique single human chromosome in a mouse background. The complete library will represent the entire human karyotype.

Selective transfer of individual human chromosomes to recipient cells

1985 ◽  
Vol 5 (1) ◽  
pp. 140-146
Author(s):  
P J Saxon ◽  
E S Srivatsan ◽  
G V Leipzig ◽  
J H Sameshima ◽  
E J Stanbridge
Keyword(s):  
Hypoxanthine Phosphoribosyl Transferase ◽  
Human Chromosomes ◽  
Isoenzyme Analysis ◽  
Phosphoribosyl Transferase ◽  
Microcell Hybrid ◽  
Multiple Copies ◽  
G 11 ◽  
Hybrid Clones ◽  
Individual Human

Two hypoxanthine phosphoribosyltransferase-deficient human cell lines, D98/AH-2 and HT1080-6TG, were stably transfected with pSV2 gpt, a plasmid containing the selectable marker Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco gpt). Hypoxanthine-aminopterin-thymidine-resistant transformants arose with a frequency of ca. 10(-6) and contained mostly single, but occasionally multiple, copies of the plasmid sequences. These transformants actively express the Eco gpt marker. Single chromosomes from two different HT1080 gpt transformants and one D98 gpt transformant, containing the integrated plasmid sequences, were transferred via microcell-mediated chromosome transfer to hypoxanthine phosphoribosyl transferase-deficient mouse A9 cells. The transferred human chromosomes were identified as 2, 4, and 22, by using a combination of G-11 staining, G-banding, isoenzyme analysis, and in situ hybridization. This system is being used to create a library of interspecies microcell hybrid clones, each clone containing a unique single human chromosome in a mouse background. The complete library will represent the entire human karyotype.

Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes

1987 ◽  
Vol 7 (8) ◽  
pp. 2814-2820
Author(s):  
T G Lugo ◽  
B Handelin ◽  
A M Killary ◽  
D E Housman ◽  
R E Fournier
Keyword(s):  
High Efficiency ◽  
Human Fibroblasts ◽  
Retroviral Vector ◽  
Human Chromosomes ◽  
G418 Resistance ◽  
Microcell Hybrid ◽  
Dominant Selectable Marker ◽  
Hybrid Clones ◽  
Microcell Fusion

We sought an efficient means to introduce specific human chromosomes into stable interspecific hybrid cells for applications in gene mapping and studies of gene regulation. A defective amphotropic retrovirus was used to insert the gene conferring G418 resistance (neo), a dominant selectable marker, into the chromosomes of diploid human fibroblasts, and the marked chromosomes were transferred to mouse recipient cells by microcell fusion. We recovered five microcell hybrid clones containing one or two intact human chromosomes which were identified by karyotype and marker analysis. Integration of the neo gene into a specific human chromosome in four hybrid clones was confirmed by segregation analysis or by in situ hybridization. We recovered four different human chromosomes into which the G418 resistance gene had integrated: human chromosomes 11, 14, 20, and 21. The high efficiency of retroviral vector transformation makes it possible to insert selectable markers into any mammalian chromosomes of interest.

Visualization of sporopollenin-containing pathogenic green micro-alga Prototheca wickerhamii by fluorescent in situ hybridization (FISH)

2009 ◽  
Vol 55 (4) ◽  
pp. 465-472 ◽  
Author(s):  
Ryohei Ueno
Keyword(s):  
Cell Wall ◽  
In Situ Hybridization ◽  
Cell Walls ◽  
Fluorescent In Situ Hybridization ◽  
Rapid Identification ◽  
Oligonucleotide Probes ◽  
Logarithmic Growth Phase ◽  
Prototheca Wickerhamii ◽  
Algal Genus

Fluorescent in situ hybridization (FISH) using taxon-specific, rRNA-targeted oligonucleotide probes is one of the most powerful tools for the rapid identification of harmful microorganisms. However, eukaryotic algal cells do not always allow FISH probes to permeate over their cell walls. Members of the pathogenic micro-algal genus Prototheca are characterized by their distinctive cell-wall component, sporopollenin, an extremely tough biopolymer that resists acid and alkaline hydrolysis, enzyme attack, and acetolysis. To our knowledge, there has been no report of the successful permeation by the oligonucleotide probes over the cell walls of unicellular green micro-algae, which contain sporopollenin. The DNA probes passed through the cell wall of Prototheca wickerhamii after treating the algal cells with cetyltrimethylammonium bromide (CTAB). Most cells in the middle logarithmic growth phase culture fluoresced when hybridized with the rRNA-targeted universal probe for eukaryotes, though individual cells included in this culture differed in the level of cell-wall vulnerability to attack by the polysaccharide-degrading enzyme, thus reflecting the different stages of the life cycle. This is the first report regarding the visualization of sporopollenin-containing, green micro-algal cells by FISH.

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