B6;D2-Tg(CAG-GFP,-Uprt)985Cdoe/J

Stock number: 021469

Description

This transgenic floxed strain enables thiouracil (TU) tagging, a method developed for isolating RNA from specific cell types from within intact tissues without the need for cell dissociation.

Detailed description

Thiouracil (TU) tagging is a method developed for isolating RNA from specific cell types from within intact tissues without the need for cell dissociation.

In this transgenic strain, a ubiquitous chicken β-actin-CMV (CAG) promoter drives expression of a loxP-GFP-3xstop-loxP cassette followed by a hemagglutinin (HA) epitope-tagged uracil phosphoribosyltransferase (UPRT) gene. Embryonic day 12.5 (E12.5) embryos and postnatal day 6 (P6) floxed animals show a widespread pattern of GFP fluorescence. The GFP-stop cassette effectively prevents any leaky read-through of UPRT expression from the floxed allele. Cre-induced excision of the floxed GFP-stop permits tissue-specific spatial expression of UPRT. Subsequent subcutaneous injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT⁺ cells exposed to 4TU produce thio-RNA which can then be purified for gene expression analysis via next-generation RNA sequencing (RNA-seq). TU tagging has been shown to have neglible effect on gene expression in cell lines and ubiquitous expression of UPRT has no effect on viability in mice.

When crossed with Tie2-cre (Tek-cre) animals, double-transgenic six-day-old mice show robust UPRT expression in PECAM⁺ (CD31) endothelial cells of the cerebellum and all other regions of the brain (e.g cortex, dentate gyrus, midbrain, choroid plexus, and hypothalamus) as well as Tie2-cre-expressing endothelial cells of the heart. Expression of UPRT is also seen in embryonic day 11.5 (E11.5) brain and heart.

When the floxed interval is excised by Math1-cre (Atoh-cre), mice show robust expression of UPRT in cerebellar granule neuron precursors (GNPs) of the postnatal day 6 brain.

Transplantation of UPRT⁺ cells can be carried out to create chimeric mice, useful in hematopoietic studies of cell migration and morphogenesis. TU tagging is complementary to cell isolation methods such as FACS panning and laser capture, with the added refinement of isolating only nascent RNA.

Development

A CAG promoter composed of chicken β-actin promoter and cytomegalovirus (CMV) early enhancer element drives expression of a loxP-GFP-3xSV40-loxP cassette followed by hemagglutinin (HA) epitope-tagged uracil phosphoribosyltransferase (UPRT) gene from Toxoplasma gondii. The transgenic vector was injected into B6D2F1 hybrid oocytes. Resulting mice were backcrossed to C57BL/6 for 4 generations by the donating laboratory (see SNP note below).

A 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. Three the 43 markers throughout the genome were still segregating with DBA suggesting an incomplete backcross.

Allele

Symbol: Tg(CAG-GFP,-Uprt)985Cdoe
Name: transgene insertion 985, Chris Q Doe
MGI accession ID: MGI:5462063
Generation method: Transgenic
Attributes: Reporter; Inserted expressed sequence
Marker symbol: Tg(CAG-GFP,-Uprt)985Cdoe
Marker name: transgene insertion 985, Chris Q Doe
Chromosome: UN
Strain of origin: (C57BL/6 x DBA/2)F1
Expressed genes: Uprt,uracil phosphoribosyltransferase,mouse, laboratory; GFP,Green Fluorescent Protein,
Site of expression: Cre-induced excision of the floxed GFP-stop permits tissue-specific spatial expression of Uprt.

For example, when crossed with Tek-cre mice, Uprt is expressed in Pecam1 endothelial cells of the cerebellum and all other regions of the as well as Tek-cre-expressing endothelial cells of the heart. Expression of Uprt is also seen in embryonic day 11.5 brain and heart.

When the floxed interval is excised by Atoh-cre, Uprt is expressed in cerebellar granule neuron precursors of the postnatal day 6 brain.
Molecular note: A CAG promoter composed of chicken beta-actin promoter and cytomegalovirus (CMV) early enhancer element drives expression of a loxP-GFP-3xSV40-loxP cassette followed by hemagglutinin (HA) epitope-tagged Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) gene. Line 985 was generated.