Initiating Genetic Aberrations in Tumourigenesis

<div><p>(A) According to the two-hit model, the first hit at the rate-limiting tumour suppressor gene provides no selective advantage for the cell. Only after the loss of the second allele of this gene is tumour formation initiated. Extra genetic changes are needed for complete transformation of the cell.</p> <p>(B) In a haploinsufficient mechanism, the first hit on the rate-limiting tumour suppressor gene already provides the cell with sufficient selective advantage to initiate tumour formation. Further events are necessary for complete transformation. These events might or might not include the loss of the wild-type allele of the rate-limiting tumour suppressor gene.</p></div

Primers and pDONR vectors used in the generation of pENTR vectors.

<p>Primers and pDONR vectors used in the generation of pENTR vectors.</p

Generation of pMULTIrec.

<p><b>A.</b> Four multisite Gateway compatible fragments (eGFPCre; IRES; puro^R and pA) were PCR amplified and cloned into the appropriate pDONR vector to make four pENTR vectors. <b>B.</b> The four pENTR clones were combined into one four-fragment Gateway clone. <b>C.</b> The four-fragment cassette was moved to pDONR/Zeo to change antibiotic resistance and subsequently sub-cloned to a vector carrying a Gateway ENTR cassette upstream of a dual selection (Neo/Kan) cassette. <b>D.</b> The resulting pMULTIrec vector.</p

The <i>Six2</i>^+/GCiP allele based on the pMULTIrec system.

<p><b>A.</b> Retrival of the <i>Six2</i>-GCIP targeting vector. <b>B.</b> Targeting of the <i>Six2</i> locus. <b>C.</b> Confirmation of correct targeting of the <i>Six2</i> locus (restriction enzyme and probe indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062054#pone-0062054-g004" target="_blank">figure 4B</a>). <b>D.. </b><i>Six2</i>^+/GCiP<i>Rosa26</i>^tdRFP kidney in culture showing GFP and RFP signals. <b>E.. </b><i>Six2</i>^+/GCiP<i>Rosa26</i>^tdRFP kidney in culture showing GFP and RFP signals and Cdh1 antibody staining. CM: cap mesenchyme; UB: ureteric bud; N: nephron.</p

The <i>Six2</i>-GCiP BAC construct made using pMULTIrec.

<p><b>A.</b> The four-fragment and dual selection cassette from pMULTIrec was cloned in a <i>Six2</i> containing BAC replacing the start codon. <b>B.</b> GFP imaging of a E13.5 embryo carrying the <i>Six2</i>-GCiP BAC. <b>C.</b> GFP imaging of whole mount E13.5 kidneys. <b>D.</b> E13.5 <i>Six2</i>-GCiP BAC kidneys cultured for four days showing GFP expression in the cap mesenchyme and Cdh1 expression in the ureteric bud.</p

Efficiency of the Gateway reactions used.

<p>Efficiency of the Gateway reactions used.</p

The <i>Nanog</i>-KiP BAC made from a modified pMULTIrec vector.

<p><b>A.</b> Generation of the <i>Nanog</i>-KiP BAC. <b>B, C.</b> mKate2 (B) and brightfield signals of iPS clones derived from <i>Nanog</i>-KiP BAC MEFs. <b>D, E.</b> mKate2 (B) and brightfield signals of iPS clones derived from wild type MEFs.</p

Efficiency of the recombineering reactions used.

<p>Efficiency of the recombineering reactions used.</p

<i>Fucci2a:</i> A bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice

<div><p>Markers of cell cycle stage allow estimation of cell cycle dynamics in cell culture and during embryonic development. The Fucci system incorporates genetically encoded probes that highlight G1 and S/G2/M phases of the cell cycle allowing live imaging. However the available mouse models that incorporate Fucci are beset by problems with transgene inactivation, varying expression level, lack of conditional potential and/or the need to maintain separate transgenes—there is no transgenic mouse model that solves all these problems. To address these shortfalls we re-engineered the Fucci system to create 2 bicistronic Fucci variants incorporating both probes fused using the <i>Thosea asigna</i> virus 2A (T2A) self cleaving peptide. We characterize these variants in stable 3T3 cell lines. One of the variants (termed Fucci2a) faithfully recapitulated the nuclear localization and cell cycle stage specific florescence of the original Fucci system. We go on to develop a conditional mouse allele (<i>R26Fucci2aR</i>) carefully designed for high, inducible, ubiquitous expression allowing investigation of cell cycle status in single cell lineages within the developing embryo. We demonstrate the utility of <i>R26Fucci2aR</i> for live imaging by using high resolution confocal microscopy of <i>ex vivo</i> lung, kidney and neural crest development. Using our 3T3 system we describe and validate a method to estimate cell cycle times from relatively short time-lapse sequences that we then apply to our neural crest data. The Fucci2a system and the <i>R26Fucci2aR</i> mouse model are compelling new tools for the investigation of cell cycle dynamics in cell culture and during mouse embryonic development.</p></div