Trinucleotide Repeats by Unknown

Author:Unknown
Language: eng
Format: epub
ISBN: 9781493997848
Publisher: Springer New York

We constructed three FMR-HyTK cassettes with various numbers of repeats: (CGG)0—control, (CGG)53—intermediate length, and (CGG)153—premutation length. They were then integrated into the RL5 locus located between the Tal1 and Pdzk1ip1 genes on chromosome 4 in murine erythroid leukemia (MEL) cell line [17] (Fig. 2). To this end, we utilized a cell line carrying a eGFP gene flanked by inverted loxP sites at the RL5 site [18]. eGFP was exchanged with loxP-flanked FMR1-(CGG)n-HyTK cassettes via transient Cre-recombinase expression. The RL5 locus was chosen to avert transgene silencing, which commonly happens with transgenes in cultured mammalian cells. The unique feature of this locus is that it is constitutively active transcriptionally, thus, a transgene integrated into it sustains its expression for many months in culture [18]. This feature of the RL5 locus was central to our experimental strategy, since spontaneous silencing of our selectable cassette could create a high background of ganciclovir resistant (GcR) clones, which would prevent us from detecting repeat-mediated events.

Fig. 2Integration of a unique copy of the FMR1-CGGn-HyTK cassette into MEL cells. The FMR1-CGGn-HyTK cassette was integrated into the RL5 locus in MEL cells via Cre-loxP recombination by replacing the eGFP gene. Two orientations of the cassette in this locus are designated as A and B

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