Fig 1.
Scheme of the MXS-chaining strategy.
(A) MluI (M), XhoI (X) and SalI (S) recognition sites are highlighted in green, blue and red, respectively. SalI- and XhoI-overhangs form a compatible pair whose cohesive ends give rise to a translatable scar (coding for valine and glutamic acid) upon ligation. Individual building blocks can be chained together in the desired order, with the MS-digested building block forming the 5’-part and the MX-digested building block the 3’-part of the new chaining module. As the XS-Scar is not cut by any of the restriction enzymes used in the chaining system the original pattern of restriction sites is recreated, which allows the reiteration of the chaining procedure. (B) New building blocks can be isolated from plasmid, genomic or cDNA by PCR. The melting temperature (Tm) of the template binding part of pairs of primers should not differ by more than 1K and be close to 60°C. 5’-extensions on forward and reverse primers enable direct subcloning of the PCR product into the MXS-chaining context. For protein coding sequences, the maintenance of the open reading frame has to be assured and the potential addition of a Kozak sequence should be considered. (C) Small building block sequences (< 120 bp) can be directly cloned from annealed complementary oligonucleotides with MluI- and SalI-overhangs. (D) MXS-chaining allows to quickly generate building blocks harboring any desired number of repeats of a specific sequence motif. This feature is of particular interest for the generation of transcriptional reporters as the number of incorporated transcription factor binding sites can be readily optimized. (E) Several chaining steps needed for the completion of a construct can be performed in parallel. Hierarchical assembly trees can be designed in such a way that intermediates yield functional subunits which can be reused for later constructs.
Table 1.
MXS-building blocks available from Addgene.
Table 2.
Vectors compatible with the MXS-chaining strategy available from Addgene.
Table 3.
MXS-cassettes available from Addgene.
Fig 2.
Efficiency of the MXS-chaining approach.
(A) Cloning efficiency of the MXS-chaining approach measured as the fraction of positive recombinant plasmids resulting from MXS-chaining ligation reactions. (B) Dependence of the cloning efficiency on sizes of MX-digested vectors and MS-digested inserts. Data from 400 different MXS-chaining ligation reactions is shown.
Fig 3.
Tagging subcellular structures in live cells.
(A) Scheme of the construct to label nuclei, the membranes and cytoskeleton components. CMV-promoters (yellow boxes) drive the expression of trimerized fluorescent proteins fused to histone 2B (H2B), Lyn-tag, β-Actin or α-Tubulin. Gray boxes are bovine growth hormone polyAs (bGHpAs). Scale bar: 1 kb. (B) Confocal section of HeLa cells expressing the construct shown in (A). Cell organelles (nucleus and membrane) and cytoskeleton components (Actin and Tubulin) are labeled. Scale bar: 10 μm. (C) Scheme of the construct. CMV-promoters (yellow boxes) drive the expression of GTS-Citrine, Lyn-3xCerulean, NLS-3xTagBFP, MTS-AzamiGreen, iRFP670-PTS1 and ER-mCherry. Gray boxes are bGHpAs. GTS: Golgi-targeting signal, NLS: nuclear localization signal, MTS: mitochondrial targeting signal, PTS1: peroxisomal targeting signal 1, ER: N-terminal calreticulin signal peptide and C-terminal retention signal -KDEL. Scale bar: 1 kb. (D) Confocal section of HeLa cells expressing the construct shown in (C). Organelles labeled are: Golgi apparatus (Citrine), membrane (Cerulean), nucleus (TagBFP), mitochondria (AzamiGreen), peroxisomes (iRFP670) and endoplasmic reticulum (mCherry). Scale bar: 10 μm. (E) Scheme of the construct. A CMV-promoter (small yellow box) drives the expression of H2B-Kaede and a CAG-promoter (large yellow box) drives the expression of Lyn-2xiRFP670. Small gray boxes are bGHpAs, large gray boxes β-Globin polyA (β-GpA) sequences. Scale bar: 1 kb. (F) Confocal section of HeLa cells expressing the construct shown in (E) before (top row) and after (bottom row) photoconversion by a 405 nm laser. The chromophore group structures in the green- and red-emitting states are depicted on the right (adapted from [49]). Scale bar: 10 μm.
Table 4.
Annealed pairs of oligos for direct MXS-chaining.
Fig 4.
Adapting the FUCCI system to mouse embryonic stem cells.
(A) FUCCI labels cells in G1 phase red, cells at the transition to S phase orange and cells in late S, G2 and M phases in green. (B) Molecular mechanism behind FUCCI. A double-negative feedback loop between the SCFSkp2 and APCCdh1 complexes exhibits bistability and leads to the oscillating accumulation of their substrates Cdt1 in G1 phase and Geminin in late S, G2 and M phases (adapted from [5]). (C) Scheme of the FUCCI-based construct to monitor cell cycle progression in mESCs. Top: A bidirectional PGK-promoter containing five enhancer elements drives the expression of Citrine-Gem(aa 1–110) and mCherry-hCdt1(aa 30–120). Small gray boxes are bGHpAs, large gray boxes β-GpAs. Scale bar: 1 kb. Bottom: Orange and purple boxes indicate the identity of the building blocks, which were assembled head-to-head into the SMX_DEST vector, see as well (D). (D) Scheme of the cloning strategy using the SMX_DEST vector to arrange building blocks head-to-head. (E) Flow cytometry analysis of mCherry and Citrine fluorescence in a clonal transgenic mESC line stably expressing the construct shown in (C). The percentage of cells in each gate is shown. (F) Analysis of the cellular DNA content. DAPI signal distribution of each subpopulation outlined in (E) and of the total culture is shown. (G) Live imaging of a clonal transgenic culture expressing the construct shown in (C). Maximum intensity projections of confocal sections are displayed. Scale bar: 50 μm.
Fig 5.
Varying the number of tetO sequences to titrate doxycycline-inducible transgene expression.
(A) Scheme of the reporter constructs containing 0–10 tetO elements and the construct expressing the reverse tetracycline transcactivator (rtTA). Scale bar: 1 kb. (B) Distribution of reporter signal in uninduced (no doxycycline added) HeLa cells measured by flow cytometry. Lines are color-coded according to the legend depicted in (A). (C) Distribution of reporter signal in induced (doxycycline added 24 h before analysis) HeLa cells. (D) Distribution of reporter signal in induced HeLa cells normalized by the background levels for each reporter construct. (E) Box plot showing the normalized reporter signal for each reporter construct.