CHAPTER 3 Results
3.2 The degree of apoptosis triggered by hBax-β, cleaved-caspase-3 and cleaved-
cleaved-capsepase-7
To further know whether toxic gene(s) can trigger the epithelial cells death or not, we chose to test three well-known genes in the apoptotic pathway, human Bcl-2–associated X protein hBax (hBax-β) and two caspase family (caspase-3 and caspase-7), as our candidate genes. Both caspase-3 and caspase-7 are truncated by
removing their pro-domains. According to previous study, truncation mutant of caspase-7 at amino acid 57 can kill cells more effectively then other mutants.[18]
(Fig.3) Therefore, in this study, we test whether the expression of hBax-β, truncated caspase-3 and truncated caspase-7 can be used for killing cancer cells.
First, three toxic genes were constructed into the expression plasmid with
bi-directional CMV (constitutive) promoter, pBI-CMV1, with EGFP simultaneously expressed as the marker for transfection. (Fig.5A) Result shows that constitutive ex-pression of hBax-β caused significant killing in both HeLa cells and 293T cells.
(Fig.5B) The apoptotic cells were blebbing with no EGFP detected. We can observe same degree of apoptosis with truncated caspase-7. We did not see a difference be-tween with or without miR-142 MBS for hBax-β and truncated-caspase-7. This is expected, as miR-142 are of low expression in HeLa cells and 293T cells.[27]
To further enhance the killing ability, we cotransfected the hBax-β and
trun-cated-caspase-7 (both in pBI-CMV1 plasmid). As there were already significant death for hBax-β and truncated-caspase-7 alone in HeLa and 293T, we did not see a visible increase in death in combination of these two. Combination experiments can be per-formed in death-resistant cells, e.g. A549. In conclusion, overexpression of hBax-β can effectively trigger apoptosis, and is therefore chosen as the toxic gene in later
experiments.
3.3 Activation of complete circuit in HeLa cells
Next, we tested the complete circuit (Figure 1B) in HeLa cells. Since in the Tet-On system, the inducer, Doxycycline, switches on the circuit, the first thing to confirm
is whether the inducer can regulate the system. We cotransfected the Tet-On 3G pro-tein driven by 342CBP and hBax-β driven by pTRE3G-BI into HeLa cells, and the result shows that this system can be controlled by Tet-On system (Fig 6). The
hBax-β gene was expressed when the inducer was present, as evident from the amount of cell death and EGFP, with or without miR-142 MBS. Moreover, we confirmed the result with time-lapse image (Figure 7). The first picture was taken immediately after the addition of doxycycline into the medium. It is evident that the circuit was activated in 30 min, as reported by both the mCherry and EGFP signal. Nevertheless, only a
proportion of the fluorescent cells rounded up (a feature of apoptosis). What’s more, the morphology of apoptosis was displayed 2 hour after the addition of doxycycline.
Most of cells successfully underwent cell death in 24 hours. However, there were still some EFGP+ cells alive, which means that cancer cells with higher death threshold (i.e. more resistant) still have the chance for escaping our circuit.
Subsequently, the other two toxic genes mentioned above were also constructed in the system to compare the degree of apoptosis. Quantification of apoptosis for all circuits is shown in Figure 8. Taken together, our results show that the genetic circuit has the ability to activate apoptosis in HeLa cells, and the expression of hBax-β trig-gered by TetOn system appears to be the best choice.
3.4 Insulator between two promoters
The ultimate question for this study is whether the miR-142 expression in hematopoietic cell lines, such as HL-60 and Jurkat, can repress the toxic signaling in our circuit. We attempt to address this, however, the problem we met is that most suspension cells were killed by electroporation in cotransfection of our circuit. Also,
the efficiency of cotransfection is very low. To solve this problem, we plan to first build stable cell line of TetOn-3G protein driven by 342CBP and constitutively ex-pressing the mCherry. This cell line (mCherry+ cells) can be selected with flow cy-tometry. Then, the line can be used to transfect the plasmid with the expression of hBax-β driven by TRE promoter, using EGFP as the marker for transfection, or
an-other selection for stable cell lines.
Nevertheless, another problem appeared in this design. The pTRE3G-Bi plas-mid we used cannot express EGFP without doxycycline. The addition of doxycycline also turns on the expression of hBax-β that leads to cell death. In other words, we cannot use EGFP+ to select for stable cells with this second layer of expression.
There-fore, we aim to create a new vector, containing a constitutive promoter and an induc-ible promoter, to express EGFP without toxic signaling. Using pBI-CMV1 as the
initial vector of our new vector (pTRE-CMV), we constructed a co-expression cas-sette with mCherry under the control of CMV promoter and the EGFP under the con-trol of TRE promoter (Fig. 9)[28]. However, the EGFP signal was present without doxycycline, suggesting that there might be interference between the constitutive CMV and the inducible TRE promoter (Fig. 11A)
To diminish the interference between the two promoters, we chose to test a previously characterized insulator, the DNA sequences at the 5’ end of the chicken β-globin HS4 (cHS4) locus in chicken’s chromosome, to prevent the unexpected signal emanating from their surrounding environment.[24, 25, 29] (Fig.10) We compared the effect of inserting zero, one and two copies of the cHS4 insulator and found that the
cHS4 core element can dose-dependently decrease the interference between the two promoters (i.e. influence from CMV promoter). (Fig.11). Quantifying the expression level of two reporters, the mCherry triggered by CMV promoter shows no different after adding the doxycycline. (Fig.12A) Insulator significantly weaken EGFP expres-sion, and it can be recover after inducer exist.(Fig.12B,12C) In this result, the
trans-fected HeLa cells are observed the lower fluorescent expression level with insulator
than without it. Moreover, we quantify the mCherry triggered by CMV promoter and
EGFP triggered by TRE promoter. mCherry shows same degree no matter the doxycy-cline exist or not, which means the constitutive promoter doesn’t effect by TRE
pro-moter. On the other hand, the expression level of EGFP less about 8-fold of construct without insulator. Then, cultured in the doxycycline-containing medium, the EGFP with insulator is recover the expression level like the no insulator one. Taken together, the insulator actually has the ability of stopping the leakage effect happened of two
promoters.
Taken together, the insulator actually has the ability to block the activation of
two promoters and CMV promoter still can constitutively without effected by insula-tor. TRE promoter can reach the same expression level of EGFP in the medium con-taining doxycycline.
Chapter 4 Discussion and Future Work
This study is inspired by a previous study that uses a customizable set of endogenous microRNAs as the multi-input sensor to achieve selective targeting of HeLa cell among the other cell lines [8]. The synthetic regulatory genetic circuit, called a cell-type
“clas-sifier”, is consisted of several miRNA binding site and LacI-controlled promoter CAGop as the upstream sensors. It is a promising platform that can be a novel anti-cancer treat-ment by using the artificial DNA constructs to specifically turn on the output when cer-tain conditions are matched.
Compared with our design, we only chose one endogenous miRNA (miR-142;
hematopoietic-high) marker and Tet-On system controlled by cell cycle regulatory pro-moter as our sensors to distinguish the dividing hematopoietic cell lines form non-hem-atopoietic cell lines. Whether miR-142 is selective enough is still an open question. Also, some cells transfected with the complete circuit (Fig 6; EGFP+ cells) can still escape
death upon induction. Hence, we also plan to simultaneously express two toxic genes linked by a “self-cleaving” P2A peptide [30, 31] to enhance the killing and to avoid the interference since the bi-directional of vector.
Another problem we encountered was the transfection efficiency of hematopoietic cells. These suspension cells are too difficult to transfect by either lipofectamine or
elec-mCherry+), however, the number of cells that survived the electroporation was too low to do further experiments, especially in this case that we want to compare the degree of cell death (data not shown). We also have attempted to generate stable 342CBP_Te-tOn+mCherry-expressing HL-60 and Jurkat cell lines with transient transfection. Unfor-tunately, in that one experiment, transfected cells were very sick and could not survive
for more than one week. We plan to use lentivirus to solve this problem in the near future.
Chapter 5 Figures
Fig1. The diagram of conception and basic design
As our mainly idea of the specificly selectivity gene circuit apporched by synthestic biology, whetehr the cell division initiated and selection marker exist are the most impotaint characteristics. (A) Schematics of a AND gate between two sensors. (B) An
illustration of actral setup in DNA seqence. hBax-β, an example of output. (C)Selectivity of circuit in different cell line.
A
B
C
Fig.2 The flow chat with fusing 4 repeats miR-142 microRNA binding site.
The sequnces of miR-142 are seperaed into two pairs of pre-annealed oligonucleotides
which contain one sticky end and one blunt end of restrcition enzyme at P1 and P4 sequences (BglII and EcoRV). By using the T4 PNK to transfer the γ-phosphate from ATP to the 5’-OH group, miR-142 MBS can ligate with vector to create a new recombi-nation vector.
Fig.3 The aligements of procaspase-3 and cleaved caspase-3 ; procasepase-7 and
truncated caspase-7.
To accelerate the protein maturation, the prodomain of both caspase-3 and caspase-7 is cleavaged initialy. Morever, we chose the truncated c-caspase-7 mutated at amino acid 57, is reported that triggers apoptosis frequenly than other mutant type. (A) The procaspase-3 gene is cloned form whole genome in the DH5α strain. Compared with the DNA sequence reported online, we construct the activated caspase -3 by removed the
prodomain. (B) To construct the truncated caspase-7, the prodomain is removed first.
Refer to the supporting information from previous study, amino acids 57-303 of caspase-7 shows the morphologies of apoptosis after activated and causes the significant cell A
B
death than others mutant. Hence, we design the primers which can bind on caspase-7 sequence started form amino acid 57 to remove the amino acid 1-56.
A
B
Fig.4 The expression level of reporter triggered by full length (949) or truncated
(342) cyclin B1 promoters at G0 and G2 phase
The first sensor is chosen because the abnormal division and unlimited proliferation are the most common feature in cancer cells. We sythcrozined the transfected HeLa cells by double thymidine assay and relased for 2 hours that allows cells to go into G2 phase. As the control group, we also block cells into G0 phase, let cells have no ability to activate the cell cycle by cultured in certain medium which is lack of nutrients for 72 hours.
Obseved the fluorescence by microscope, the reporter (mCherry) triggered by (A) truncated cyclin B1 promoter (342 CBP) and (B) full length cyclin B1 promoter (949CBP) shows significantly high expression in G2 phase than G0 phase. (C) The percentage of fluorescent-positive cells indicate that the 342 CBP can successfully
regulate the gene accroding the cell cycle.
C
B A
HeLa cells 293T cells
Fig.5 The transfected cancer cells which constitutively express the toxic gene results
in cell apoptosis in HeLa cells and 293T cells.
(A) The illustration of constructs containing different toxic gene and with or without miR-142-5p MBS located at the downstream triggered by CMV promoter. The EGFP reporter is used as transfected maker. (B) The image of cell morphology result that the overexpression of hBax-β gene and truncated caspase-7 gene can efficiently killing the cancer cells. The transfected cells expressing cleavage caspase-3 gene
show the EGFP and caused less cell death after transfected for 24 hours, which means that overexpression the cleavage caspase-3 alone doesn’t have the enough ability of killing the cancer cells. We also co-transfect the two toxic genes to know whether the killing ability is more powerful than expressed alone or not. In this re-sults, the cleavage caspase-3 and truncated caspase-7 shows the significant killing
co-work with hBax-β gene.
Fig.6 The inducible promoter triggers the toxic gene results in cell apoptosis.
The circuit is containing the tetOn-3G system regulated by 342 CBP and toxic gene activated by TRE promoter. If the Dox exist, the toxic proteins are produced and cause the cell death. This result explains the same conclusion of the data mention above (Fig.5B). We consider the hBax-β gene shows the most powerful killing than others and successfully induced the apoptosis happened. Control: transfected 342CBP_TetOn +
mCherry and pTRE_EGFP. Positive control: transfected 342CBP_TetOn + mCherry and pTRE_EGFP (treated with STLC for 24 hours)
Fig.7 Time lapse image of HeLa cells transfected pTRE3G-BI_ hBax-β+EGFP after
inducing for 24 hours by doxycycline.
As this result, the HeLa cells start producing the hBax-β and EGFP in just 30 minutes after inducing by Dox. After 24 hours, most positive-transfected cells round up and
blebbing that means that cells are going to apoptosis.
Fig.8 Measurement the cell viability and cell death rate by trypan blue exclusion
assay in HeLa cells.
To measure the cell survival rate or death rate , we stain the transfected cells with try-pan blue and calculate the living cells and death cell by Hemocytometer. All the cells are co-transfected with 342CBP-TetOn + mCherry and toxic gene derived by
pTRE3G-BI.( A) Cell viability. (B) cell death rate. This data shows that most of the living cells number are decreased except the pTRE_EFGFP + c-caspase3::miR-142-5p MBS. pTRE_EGFP indicate the survival cells and pTRE_EGFP(STLC) indicate the cell death control. ns, non-siginificant(P > 0.05);**,P ≤ 0.01;***, P ≤ 0.001;**** P ≤
0.0001 (P values)
A B
(%)
Fig.9 Flow chat of cloning the construct containing constitutive and inducible
pro-moter.
In order to make a vector that can continuously produce the reporter gene and activate the toxic gen only when we add the inducer, we combinate the pBI-CMV1 and
pTRE3G-BI by digested with XhoI and XbaI. There are one side consist of CMV
en-hancer and CMV promoter and another side consist of 7X tet operator and TRE prom-ter.
Fig.10 Designs of vector and two promoters and insulator.
(A) Illustration of 2x core element of insulator located between two different promoters.
Moreover, we anticipate the cell would change the color form red to yellow after doxycycline exist, which express the red and green fluorescent at the same time. (B) The design of constructing the vector containing the insulator. We use the SalI to
ligate with XhoI since they have the same cutting site. According to this design, there A
B
is a scar happened but it doesn’t effect next enzyme digest and we can inset another core element of insulator as much as we want.
A
B
Fig.11 The blocking ability of different copies of core elements of insulator in HeLa
cells.
There are three kind of copies between two of the promoters (A) no insulator (B) 1x core insulator (C) 2x core insulator are used as our test group. No matter the inducer
exist or not, the mCherry driven by CMV promoter is produced all the time in all the groups. The EGFP driven by TRE promoter is significantly decreased in insulator-tained group than no insulator group. As this result, we can simplify learn that this con-struct can actually block the leakage between two different type of promoters.
C
Fig.12 Quantify the expression level of reporter triggered by two promoters
con-taining different copies insulator.
(A) The construct (Fig.11A) and Tet-On system controlled by 342 CBP are co-trans-fected into HeLa cells. As the result, the expression level of mCherry triggered by
CMV promoter has no change after adding the doxycycline, which means the con-stitutive promoter can stably express the downstream gene. (B) Expression level of EGFP is significantly decreased that containing one or two copies core element of
insulator. (C) Adding the doxycycline is attended, EGFP can be up to the same ex-pression level as the one without insulator. As the conclusion, the problem of leak-age can be resolved by inserted the insulator between two promoters. (n=20) ns,
non-siginificant(P > 0.05); **** P ≤ 0.0001 (P values)
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