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

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.

Both digestion systems were incubated in 37℃ for 2h

Figure 3.

Result：backbone plasmid pCDNA3.0 was fully digested，but as the fragment excised from pCDNA3.0 was too short(which means too small in quality because it has the same amount of substance as the backbone’s) in length(less than 50bps),it cannot be identified in agarose gel-electrophoresis. When digested in a least one restriction site, plasmid turns from supercoil status to linearized and has lower mobility in gel-electrophoresis, so it can only be judged that backbone was definitely complete digested in at least one restriction site. Such assessment applies also to digestion of PCR product from whom the excised parts are almost never more than 50bps.

1.4 Ligation

Ligation type：1 vector+1 insert fragment

Desired ratio of the amount of substance：

Vector：insert fragment =1:5

Ligation for 2h at room temperature(25℃)

1.5 Transformation of ligation product

Transformation system

After 2h ligation，ligation system,as well as Competent E.coli taken from -80℃ storage， was first incubated in ice for 5min before mixing together.

Transformation system continued to be incubated in ice for 30mins,42℃heat shock for 60s，then again ice incubation for 5mins.

Transformation system was added 600μL antibiotic-free LB medium and put in 37℃ thermostat shaker for 30mins-resuscitation（expression of Amp resistance protein）

Transformation system was then centrifuged at 130,000g for 3mins.All but little supernatant was kept to suspend the E.coli，which was later transferred to the LB agar plate. The plate was put in 37℃ thermostat incubator overnight for colonies growth.

1.6 Verification of the colonies（each as a single clone）

Figure 4.

colony PCR set up

Figure 5

Result：Clear band around 1000bp appeared for every 12 clone.

1.7 Plasmid extraction and verification

6 of the single-clone colonies were selected and added to 5ml Amp-LB medium and put to 37℃ thermostat shaker.

The bacteria were centrifuge after 16h-growth in the thermostat shaker.Plasmids were extracted using endotoxin-free kit.

The plasmids were later digested by BamHI and XhoI for verification.

Figure 6

Result: Clear band around 1000bp appeared. Plasmids from every 4 clones were verified.

Plasmids were later sent for sequencing

2013.6.1-6.14

Construction of pCDNA3.0-pEF1a-tTA-PGK-BSD

pCDNA3.0-pEF1a-rtTA-PGK-BSD

pCDNA3.0-pEF1a-tTA advanced-PGK-BSD

pCDNA3.0-pEF1a-eGFP-PGK-BSD

Figure.7

PCR system set up

4. Endonuclease digestion

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].

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.