UPDATE 09/18/2013

Molecular construction of the up stream elements(regulating parts,protein tTA，rtTA，tTA advanced ，and eGFP for contrast ) of tet systems.

Introduction

Team members in Charge：Dawei He (2013.5.20-9.16)and Yiming Fang(2013 5.20-7.16)

Author: Dawei He

Team members in Charge：Dawei He (2013.5.20-9.16)and Yiming Fang(2013 5.20-7.16)

Despite a good comprehension of the knowledge of Molecular biology and its relating experimental technologies，We never came to and stay in a Molecular biology lab and were completely lack of any experience. To achieve the designated tasks in extremely insufficient time(less than 3 months),We had to learn fast and start our work independently as soon as possible.

2013.5.20-5.23

Contents: Contact and learning of Molecular construction

Team members in Charge：Dawei He (2013.5.20-9.16)and Yiming Fang(2013 5.20-7.16)

1. DNA extraction techniques: Plasmid extraction, DNA clean up(extraction from PCR or restriction endonuclease digestion systems,extraction from Ag gel, etc)

2. PCR techniques:

Primer(for molecular construction or sequencing) designing, PCR for molecular construction, colony PCR for verification.

3. Endonuclease digestion

2013.5.24 to 5.31

Contents: First plasmid construction：pCDNA3.0-PGK-BSD

Annotation：pCDNA3.0 is a backbone plasmid ideal for transient expression in our designated cell lines including HEK293T,HepG2 and Bocs. All elements of our project are first to be incised into pCDNA3.0 and tested during transient expression in cell lines. Driven by promoter PGK,BSD express (!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!1)

Figure 1.Backbone plasmid pCDNA3.0(completed featured)

Figure 2.pCDNA3.0-PGK-BSD

construction details:

1.PCR of PGK-BSD(915bp in length) from donor plasmid(as PCR template)

Primers：Forward primer with restriction site BamHI and necessary base pairs from 5’ end

Reverse primer with restriction site XhoI and necessary base pairs from 5’ end

Bases pairs should appear at both flanks of restriction sites to provide anchoring sites for endonuclease,3’ end of the restriction sites in a primer is already covered by base pairs, so some base pairs must be added to its 5’ end. Without such manually added base pairs from 5’ end, efficiency of endonuclease digestion of PCR product will be significantly reduced.

1.1 PCR system set up

integration

5×fastpfu PCR buffer	5μl
2.5mM dNTP：	2.5μl
10uM PB F primer	0.8μl
10uM PB R primer	0.8μl
Template(plasmid)	4μl(5.36ng/ul,21.4ng in total. Acceptable quantity from 5-30ng)
Fastpfu DNA polymerase	0.5μl
ddH20	11.4μl(up to 25ul)

1.2 temperature program set up

Annealing temperature in main PCR cycles =Primer Tm-5℃

Pre-cycles with lower annealing temperature in PCR process of primers with restriction sites(or any primers that include bases pairs not complementary to their original templates ) are necessary to achieve both specificity and quantity demand of PCR product.

During the first PCR cycle, primer binds only and incompletely to its original templates, so tm is lower compared to the one when primer binds total- complementarily to its product generated since the first cycle, which was why the idea of adding pre-cycles before main cycles came up. Although annealing temperature in pre-cycles can be calculated to details, the temperature 15℃ below annealing temperature in main cycles will be quite universal.

Primer binding to its original template

Primer binding to its PCR product

1.3 Endonuclease digestion of backbone plasmid and insert fragment（PCR product）

PCR product PGK-BSD was purified using PCR clean up kit.

Life there and back again

In 2006, Shinya Yamanaka, a Japanese scientist who afterward shared the 2012 Nobel Prize in Physiology or Medicine with John B. Gurdon[5], proved that introduction of a small set of transcription factors into a differentiated cell was sufficient to revert the cell into a pluripotent state. In his strikingly bold experiment, what Yamakana did was introducing 24 genes encoding the transcription factors which were considered as candidates to reinstate pluripotency in somatic cells to skin fibroblasts in one step. Surprisingly, he found out that a few of these cells actually generated colonies that showed a remarkable resemblance to ES cells. Then he reduced the number of genes capable of inducing such colonies one by one, and finally, four factors (Myc, Oct3/4, Sox2 and Klf4) were picked out, whose combination were identified to be sufficient to reprogram the skin fibroblasts to pluripotent stem cells[3]. These synthetical new stem cells were thereafter named by their Japanese father as iPSCs (induced pluripotent stem cells). A year later, using the same four factor combination from the 2006 paper (Myc, Oct4, Sox2 and Klf4), Yamanka's group successfully produced human iPS cells, whereas another laboratory, James Thomson's lab, achieved the same goal with a somewhat different transcription factors combination (Lin28, Nanog, Oct4 and Sox2)[4].

A technology with many possibilities

The groundbreaking work that Yamakana and his group did in 2006 truly opened up a completely new research field to this world. It was a paradigm-shifting fundamental discovery, as it was the first demonstration that an intact differentiated somatic cell could be reprogrammed to become pluripotent. iPSCs technology, a new technology with many possibilities, attracts scientists and researchers all over the world, mainly for its three unique superiorities:

- No strong immunological reaction: Since iPS cells can be cultured from the patient's own cells, it is not quite possible to lead to immunological reaction when directionally differentiated iPSCs are transplanted after in vitro culture. While in 2011 several studies argued the weak immune reaction to transgene-free iPSCs, these arguments were subsided later by other scientists and Yamanaka himself, pointing out that "the most prominent study that reported the immunogenicity of the cells examined undifferentiated iPSCs, which will never be used in cell transplantation therapy"[6]. Recent work by Araki and Guha used syngeneic mouse models to demonstrate that transplanted iPSC-derived embryoid bodies, skin and bone marrow tissues engraft efficiently with almost no signs of rejection[6].

- "Thank god we don't need embryos anymore!": Simultaneously, the technology of iPSC was also hailed by many commentaries as a milestone advance that solved the ethical and political problems in stem cell research field. In the past, stem cell research relied heavily of embryonic stem cells harvested from embryos. Human embryos are not easy to come by, and many people consider research involving use of human embryonic stem cells to be ethically questionable[8]. With no further evidence, iPSC technology not only breaks the ethical barrier of relying on using eggs or earlier embryos for deriving stem cells, but also leads to a convenient way of obtaining patient-specific stem cells.

- Wide application in different fields: Nowadays, scientists are making progress toward applying iPSCs in many fields. One of the applications - which has already become reality - is to use iPS cells in both analysis of disease mechanisms and investigation of potential new treatments. In the future scientists hope to be able to use iPS cells to culture cells that can be transplanted into the body and replace diseased cells. Two examples are the dopamine-producing cells in the brain that degenerate in patients with Parkinson disease, and the insulin-producing cells that die off in patients with diabetes[13]. So far, more than 100 reports published in the past three years using disease-specific iPSCs[8].

These advances above together made the iPSC to be the "star cell" which offers immense potential as a source for regenerative therapies. However, the intrinsic qualities of self renewal and pluripotency that make these cells so therapeutically promising are also responsible for an equally fundamental tumorigenic potential, and the the intrinsic shortcomings of the current methods of inducing pluripotency even pose more uncontrollable risks on that. High rates of tumorigenicity has become a most crucial hurdle for large-scale clinical implementation of iPSC technology.

Challenges

Tumorigenicity as a clinical hurdle

The risks of iPSC tumorigenicity have been widely concerned over the past several years. In the study in 2009, Yamakana tested 55 mice transplanted with SNS (secondary neurospheres) from 11 TTP-iPS cell clones, 46 mice among them died or became weak within 9 weeks after transplantation because of tumors. Whereas in contrast group, the number of the tumor-showing mice transplanted with SNS derived from the ES cell clones was only 3 among 34[11]. This result indicated that the mice transplanted with iPSCs have a much higher rate of tumorigenicity than with ESCs.

- The "fantastic four"are double-edged swords: In the initial study in 2006, Yamanaka has pointed out that among the four transcriptional factors he used, two (Oct-4 and Myc) were oncogenes[3]. This led to the result that the iPSCs with the four exogenetic oncogenes were more prone to tumorigenesis. However, subsequent studies successively substantiated that both the Oct-4 and Myc are not essentially required for cell self-renewal. For example, in making human iPS cells, Yu et al. used a different set of four factors (Oct-4, Sox-2, Nanog, and Lin-28) to induce pluripotent stem cells from human somatic cells. These results began to raise the scientists' hopes to find the better combination of the cocktails to induce the safer iPSC. However, it turned out to be wishful thinking.

Scientists figure out that whatever the combination is, almost every inducing/reprogramming factors remaining in the cocktail are oncogenes by definition. Their over-expression has be associated with some forms of cancer. Of particular importance is the MYC transcription factor, which has emerged as one of the fundamental genes shared by iPSCs and cancer. Ectopic activation of OCT4 in somatic cells, induces dysplastic development and features of malignancy. NANOG has a role in the self renewal of CD24+ cancer stem cells in hepatocellular carcinoma. SOX2 has been shown to drive cancer-cell survival and oncogenic fate in several cancer types, including squamous cell carcinomas of the lung and esophagus. Klf-4 has been reported to promote DNA repair checkpoint uncoupling and cellular proliferation in breast cancers by p53 suppression[7].

- Risks from delivery methods: Compared to ESCs, iPSCs are exposed to a number of factors that could promote oncogenic transformation, such as genomic insertion of reprogramming vectors, over expression of oncogenic transcription factors and a global hypomethylation resembling that seen in cancers[7].

Methods of diminishing the tumorigenic transformation of iPSCs have mainly focused on a variety of gene delivery vectors that minimize genomic disruption. These strategies can be generally divided into two categories: integrating vectors that can be excised from the host genome and non-integrating vectors. However, all methods can not escape their shortcomings of low transduction efficiency, or even bring about new risks. Here is a table which concludes the oncogenic risks associated with methods of inducing pluripotency in somatic cells[7].

Table 1 Oncogenic risks associated with methods of inducing pluripotency in somatic cells

Method of induction	Strengths	Weaknesses
Lentiviral vector	Robust reprogramming efficiency	Genomic integration, reactivation of integrated transgenes
Cre recombinase	Little genomic disruption	Low transduction efficiency, integration of LoxP sites into host genome
PiggyBac transposition	Minimal risk of genomic disruption	Low transduction efficiency, risk of uncontrolled rounds of excision and integration
Adenoviral vector	Low risk of genomic integration	Low transduction efficiency, limited transgene expression
Plasmid transfection	Minimal risk of genomic disruption	Very low transduction efficiency typically requires use of oncogenes such as the SV40LT antigen for successful induction of pluripotency
Minicircle	Minimal risk of genomic disruption	Low transduction efficiency
Sendai virus	Minimal risk of genomic integration ,relatively high transduction efficiency	Risk of continuous replication of viral vector in cytoplasm, leading to aberrant silencing of pluripotency transgenes
Synthetic mRNA	No risk of genomic integration, ability to control transgene expression	Variable transduction efficiencies, high technical expertise required
Protein transduction	No risk of genomic integration, ability to control transgene expression	Very low transduction efficiency, labor intensive
microRNA transfection	No risk of genomic integration	Low reprogramming efficiency
Small molecules	No risk of genomic integration	Variable off-target effects

From the table above, we can generally realize that these promising strategies for teratoma prevention mostly improve the safety of iPSC generation at the expense of efficiency, which is still another remaining challenge in iPSC technology.

In summary, it wouldn't be more perfect if scientists found a method which guaranteed both the safety and efficiency in iPSCs technology. Just as Andrew S Lee wrote on Nature Medicine, "Ideally, the most stringent safety regimes would utilize a flexible, combinatorial approach that may require tailoring for specific PSC lines or graft types. Should these techniques fail to adequately remove enough residual PSCs, retrospective tumor treatments may also be used, including oncologic chemotherapy, radiation and surgery or the incorporation of suicide ablation genes[7]." Admittedly, just as the saying goes, "You can't have your cake and eat it!" With our knowledge of iPSC biology, it is not likable that such a perfect regime would emerge in several coming years. However, as a group of young pre-scientists like us, maybe, it is our time to look into this problem and try to figure it out in another way.