Tracking Proliferative History in Lymphocyte Development with Cre-Mediated Sister Chromatid Recombination
Figure 1
Genetic labeling of proliferating cells in vitro with the Tlox design.
(A) A comparison between the Tlox and a single loxP sequences. Codons of the reading frame are marked in the Tlox sequence. The stop codon (in red) is in the overlapping region between the first and the second loxP sequences in Tlox. (B) Schematic of Tlox on MSCV retroviral backbone. In the presence of Tlox, both GFP and tdTomato are transcribed, but only GFP (indicated by the green color) is translated. When Tlox is replaced with a single loxP, both GFP and tdTomato (indicated by the orange color) are expressed. 2A is included as a target sequence for auto cleavage [34] to separate GFP and tdTomato. (C) Diagram illustrates Cre-mediated sister chromatid recombination via the Tlox site during mitosis. Results of both equal and unequal exchanges after the G2/M phase are shown. Unequal exchange (bottom part) produces daughter chromosomes carrying either single or triple loxP site. (D–E) 3T3 cells harboring the reporter construct were transduced with control empty virus or Cre virus and analyzed by fluorescent imaging (D) or FACS (E) 48 hours post viral transduction. (F) Cell counts of total and tdTomato positive cells at 24 or 48 hours post viral transduction. Non-transduced cells were excluded based on lack of hCD2 expression. Percentages of tdTomato positive cells are indicated in the plot. (G) PCR assay of genomic DNA with primers flanking the Tlox site. M, 1 kb-DNA size ladder; 1, reporter plasmid control; 2, total cells at 48 hours after Cre transduction; 3, parental cells before Cre transduction; and 4, FACS sorted double positive cells at 48 hours after transduction. Bands representing 1loxP, 2loxP, and 3loxP were verified by sequencing analysis after subcloning the PCR products into Topo vectors.