Team:SYSU-CHINA/Project/CyclebowDesign

Hwang, H.C. and B.E. Clurman, Cyclin E in normal and neoplastic cell cycles. Oncogene, 2005. 24(17): p. 2776-86.

Ohtani, K., J. DeGregori, and J.R. Nevins, Regulation of the cyclin E gene by transcription factor E2F1. Proc Natl Acad Sci U S A, 1995. 92(26): p. 12146-50.

Guo, Z.Y., et al., The elements of human cyclin D1 promoter and regulation involved. Clin Epigenetics, 2011. 2(2): p. 63-76.

Pei, D.S., et al., Analysis of human Ki-67 gene promoter and identification of the Sp1 binding sites for Ki-67 transcription. Tumour Biol, 2012. 33(1): p. 257-66.

Cyclebow design

Design Framework

      Our system consists of three circuits:STARTER CIRCUIT,COUNTER CIRCUIT and REPORTER CIRCUIT.

Figure 1. Concept map of STARTER CIRCUIT

       STARTER CIRCUIT is a tet-on system that with the induction of Doxycycline, recombinase Cre will express. The expression of Cre triggers COUNTER CIRCUIT and REPORTER CIRCUIT.

Figure 2. Concept map of COUNTER CIRCUIT

      COUNTER CIRCUIT is the recombinase circuit with lined up SNAP UNIT and recombinase UNITs.The SNAP UNIT consists of SNAP-tag (NEB pSNAP-tag®, which can be labeled by adding specific substrates) flanking with LoxP (the RTS of Cre).The recombinase UNITs include VIKA UNIT, consisting of Vika recombinase flanking with Vox (the RTS of Vika) (we intended to include more UNITs such as VCRE UNIT, but failed due to time limitation). All of the UNITs in circuit 2 contain a NLS (Nuclear localization sequence) at the beginning of the protein and a terminator at the end.

      The highlight of this circuit is its cell-cycle dependent promoter. After studying publications [10][11][12][13] about cell-cycle dependent promoters, we selected three promoters of pCyclinE (G1), pKi67(G1) and pCDK4(G1/S). Cdk4, CCNE and Ki-67 are cell proliferation markers, which tightly associate with maintenance and regulation of the cell division. These proteins are present during all active phases of the cell cycle (G1, S, G2, and mitosis), but are absent from resting cells (G0). Their promoters contain multiple transcription factor binding sites .The extracellular signals functions through the signal transduction pathways converging at the binding sites to active or inhibit the promoters activity and regulate the cell cycle progression. Different signal transduction pathways regulate the promoters at different time to get the correct cell cycle switch. In our project, we exploit the synchronism betweenthese promoters and cell cycle to regulate the expression of our target genes, which enables us to count the cell-cycles one by one.With the cell-cycle dependent promoter, genes can be only translated during the G1 state of the cell-cycle. Confirmed by our last year’s researches, the recombinase need to accumulate to a certain concentration to work, and G1 state is only long enough for one recombinase to work.

Figure 3. Concept map of REPORTER CIRCUIT

      REPORTER CIRCUIT is the florescence circuit with lined up SNAP UNIT and florescence UNITs. The SNAP UNIT is same with that in circuit 2. The florescence UNITs include mCHERRY UNIT and CYAN UNIT. The florescence UNITs are similar in design with the recombinase UNITs, replacing NLSwith degradation tag DBOX but for the CYAN UNIT(we intended to include more UNITs such as GFP UNIT, but failed due to time limitation) which is just a plain CYAN. The promoter in circuit 3 is the constitutive promoter pEF1α.

Figure 4. Concept map of how Cyclebow system works

      When three circuits are all inserted into cells, the only protein that will be express is SNAP-tag, which is imperceptible. Therefore our cells will be “grey” during their first cell-cycle. When STARTER CIRCUIT is induced and Cre is expressed, the SNAP UNIT in COUNTER CIRCUIT and REPORTER CIRCUIT will be cut out as it is flanked by same oriented LoxPs. Vika will not express right away as the promoter has to wait till G1 to function, but mCherry will express, so the cells will present red florescence. As the red colored cells proceed to their second cell-cycle, Vika will express and the VIKA UNIT as well as the mCHERRY UNIT will be cut out. Cells turn blue as CYAN begins expressing and since there is no more UNITs, cells after this will all be blue. For illustration convenience and due to time limitation, our design only contained two UNITs in COUNTER CIRCUIT, and three UNITs in REPORTER CIRCUIT, which can distinguish cells that underwent 2 cell cycles. By adding more UNITs of different recombinases and different colors, we can distinguish more.

Circuit Construction

      UNITs with tags and RTSs are constructed using PCR and oligonucleotide ligation by the following procedure. Our vector is pEntry which contains attL1 and attL2 site for recombination into eukaryotic expressing vectors.

Figure 5. Three steps to construct our UNITs using PCR and oligonucleotide ligation

      Our UNITs all contain XbaI and SpeI enzyme sites, so we can easily line up the UNITs using 3A assembly. After completion of the circuit, we recombine our circuit from pEnty into eukaryotic expressing vectors which also contain Lenti Virus parts for stable transfection. We need three circuits in total, therefore we need three eukaryotic expressing vectors with different anti-biotic resistance. We chose Puromycin, Neomycin and Hygromycin.

      Since we don’t have a eukaryotic expressing vector that contains cell-cycle dependent promoter, we have to reconstruction a vector by exchanging its constitutive promoter into our G1 state promoters.

Tests on immortalized cell lines

      Our project is closely related to the cell cycle, so confirming the cell cycle of Hela, MCF7 and A-549 cell line is vital. The method we use is: double thymidine block, collect cells every 2 hours after releasing, dye DNA with PI and run samples with the flow cytometry.

      Before constructing the circuits, we need to confirm whether the G1 state promoters we obtained has an average promoter function. We ligate a GFP after our promoters, and transient transfect the vector into 293T cells, and detect the green light. If green light can be detected, we can confirm the function of our promoters. After this, we can proceed the circuit construction, otherwise we will have to select new promoters first.

      Moreover, whether the selected G1 state promoters only function at G1 state needs to be confirmed. We construct stable cell lines of G1 state promoter vector with SNAP UNIT for the confirmation. After double thymidine block and releasing (when the majority of cells will be in the G1/S state), we block the previous SNAP tags so that they could not labelled, and collect cells at certain time points, dye with PI and SNAP-Cell 505-Star® and run samples with the flow cytometry. We expect the SNAP signal in consistence with the G1 state signal, representing that the selected G1 state promoters indeed only function at G1 state.

      Since other parts we use (recombinases, fluorescences, tags etc.) come from either our previous project or the laboratory, which are all confirmed of function, there will be no need to conduct tests on them.

      In order to gain results that has statistical significance, we decide to combine Cyclebow with thymidine block which can regulate the cells’ division activity. We intend to add Dox after the first block, so that the cells will turn red during the second block, and then turn blue after the second release. Therefore we need to confirm that the majority of cells will be blocked at G1/S after first blocking, and Dox inducing for 16 hours is enough for Cre to express. We use flow cytometry and Western Blot to confirm these two premises.

Cyclebow Construction

      OThe STARTER CIRCUIT serves as the switch of the whole system, therefore its construction deserves the priority. We use Lenti Virus to construct the stable cell lines of STARTER CIRCUIT in Hela, MCF7 and A-549 cell line. The vector of the STARTER CIRCUIT contains Neomycin resistance, so we use G418 to screen the cells. After the screening period, we add Doxycycline, and RT-PCR is conducted to confirm the expression of Cre.

      Before constructing the circuits, we need to confirm whether the G1 state promoters we obtained has an average promoter function. We ligate a GFP after our promoters, and transient transfect the vector into 293T cells, and detect the green light. If green light can be detected, we can confirm the function of our promoters. After this, we can proceed the circuit construction, otherwise we will have to select new promoters first.

      Moreover, whether the selected G1 state promoters only function at G1 state needs to be confirmed. We construct stable cell lines of G1 state promoter vector with SNAP UNIT for the confirmation. After double thymidine block and releasing (when the majority of cells will be in the G2 state), we block the previous SNAP tags so that they could not labelled, and collect cells every 2 hours, dye with PI and SNAP-Cell 505-Star® and run samples with the flow cytometry. We expect the SNAP signal in consistence with the G1 state signal, representing that the selected G1 state promoters indeed only function at G1 state.

Figure 6. Procedure to test our Cyclebow