Notebookt/Methods

Methods

Introduction

Our experiments can be divided into two parts, one is the design and construction of genes and vectors, the other is test of parts and circuits with cells, including hepatoma cells, liver cells and iPSCs. So our protocol can be clearly be divided into these two parts, one for molecular operation, the other for cellular tests. Besides, quantitive measurement, like qPCR and western-blotting, is added in the molecular operation parts because it does not need cell operation.

Molecular Operation

< In the process of constructing circuit on vectors, PCR, PCR clean up, digestion of restriction endonuclease, gel extraction, ligation, transformation, colony PCR and plasmid extraction is implemented. Besides, for quantitive measurement, qPCR and western-blotting is used..

PCR

1. Prepare the reaction mix:

5x Fastpfu buffer	10ul
2.5mM dNTPs	5ul
Forward primer	0.8ul
Reverse primer	0.8ul
Template DNA	40-50ng
FastPfu	1ul
ddH2O	add to 50ul

2. Set the program of thermal cycler

（1×）	95℃	3min
（25×）	95℃	20s
	A-5℃	20s (Tm≈A℃)
	72℃	30s for each 1kb
（1×）	72℃	5min
（1×）	4℃	forever

3. Run the PCR program.

4. After PCR, load ~2ul to a 1% agarose gel to have a quick run checking the production of desired fragment.

PCR clean up

Use Anxygen PCR clean up kit for PCR clean up. In addition, restriction endonuclease digestion mix can be cleaned if the fragment to be deserted is less than 50bp.

1. Add 150ul of buffer PCR-A, vortex.

2. Pipette the liquid to a column, wait for 1min, then centrifuge 1000xg for 1min and 12000xg 30s.

3. Use Buffer W2(700ul for the first step and 400ul again) to wash, 12000xg for 1min.

4. Dry the column on 65°C, warm the elutent in the meantime.

5. Use 25-30ul preheated elutent to wash DNA, wait for 1min and centrifuge 12000xg for 1min.

6. Use Nanodrop to measure the concentration and quality of the DNA product.

Digestion of restriction endonuclease

NEB, Fermentas and Takara restriction enzymes are used. Before use, check the proper buffer for the enzyme, if the activity is low, add twice the amount but prevent the enzyme volume more than 10%, or star activity may rise.

1. Set up the reaction mix:

DNA	1ug for insert fragment, 3ug for vector
10x buffer	2ul
Enzymes	1ul each
ddH2O	add up to 20ul

2. Carefully mix the reaction mix and put it into 37°C water bath. Control the reaction time 2. Carefully mix the reaction mix and put it into 37°C water bath. Control the reaction time .

3. Load 1.5ul mix on 1% agarose gel to have a quick run to check if the reaction is clear.

4. 75°C water bath for 5min to stop the reaction.

Gel Extraction

Use Omega Gel Extraction Kit to do gel extraction.

1. Cut the gel slice and weigh, 1ug for 1ul equal volume.

2. Add 1x sample volume of Binding Buffer(XP2), add 1x sample volume of isopropanol if DNA is less than 500bp. Add 5ul of 5M pH5.2 NaAc when necessary.(When the color of liquid is orange or red, add it until it turns light yellow)

3. Incubate the mixture in 65°C for 7min or until totally dissolved. Vortex every 2-3min helps.

4. Put it in room temperature to let it cool down to some extent(control the temperature before the liquid become solid). Load it into a column(for more than 700ul, load it in several times). Centrifuge 1000xg for 1min then 10000xg for 30s.

5. Add 300ul Binding Buffer to wash the column, 10000xg 1min.

6. Wash with buffer SPW, add 700ul, wait 2-3min and centrifuge 10000xg. Repeat 1 time.

7. Discard the liquid and centrifuge 10000xg 2-3min to dry.

8. 65°C dry heat for elutent and column for totally dry. Wash it by 30-50ul elutent, centrifuge for 1min.

9. Use Nanodrop to measure the concentration and quality of the DNA product.

Use Fermentas T4 ligase for ligation.

Vector(about 5000bp)	40ug
Insert fragment	5x chemical amount of vector
T4 ligase buffer	3ul
T4 ligase	0.3-0.8ul
ddH2O	add up to 30ul

For overnight ligation, use 0.3ul ligase, and for ligation that only proceeds in 2 hour, use 0.8ul ligase. The reaction temperature is room temperature, or a little bit lower than room temperature.(16°C)

Transformation

1. Add 200ul TCM to whole reaction mix(for ligation mix or digestion mix), put on ice for 3-5min. This step is optional when plasmid is transformed.

2. Add 50ul chemical competent cells（Top10）and mix gently. Incubate on ice for 30 min

3. Heat-shock in a 42℃ bath for 60 sec. Put it on ice for 3min.

4. Add 0.5 ml LB/SOC(antibiotic free) medium and incubate at 37.0C for 30-60 min

5. Spin at 8,000*g for 3min. Decant most of LB medium but leave –100ul behind to resuspend the bug.

6. Plate all of the bug suspensions onto one LB(Amp+) plate. Mark the name of the plate and put it into 37°C incubator for 14-18h.

Colony PCR

Just like PCR protocol, but use takara rTaq enzyme.

Plasmid Extraction

Use Omega Endo-free kit for the plasmids for cell.

Use Anxygen Plasmid miniprep kit for plasmids for molecular cloning.

Follow the protocals provided in the kit. But a 65°C elutent can cause higher efficiency.

Cellular tests

In the process of cellular tests, transient transfection, virus package, selecting stable cell line are included.

Transient transfection

1. Seed cells a night before.

2. Refresh medium right before transduction. Cell density should be around 60% at the time of transduction.

3. For a single well in a 12-well plate, dilute 1.5ug plasmid DNA in 100ul opti-MEM and then add 4ul PEI.

4. Gently mix and then incubate for 20min.

5. Add the mixture to cells and refresh after 6-8h.

6. 1:1000 doxycycline is used to induce the Tet system.

Virus packaging

1. seed cells a night before.

2. refresh medium right before transduction. Cell density should be 40% at the time of transduction.

3. For a single well in a 6-well plate, dilute totally 3ug plasmids in 100 ul CaCl2,gently mix.We use PCGP/VSVG to package retrovirus and PSPAX2/MD2G to package lentivirus.

4. Add the mixture to 100ul BBS carefully and incubate for 20min.

5. Add the mixture to the cells and refresh after 6-8h.

6. collect virus after 48h and store in -80°C.

7. For condensed virus, use 10cm culture medium and proportionally increase the reagents as described above. Medium containing virus is sealed by mineral oil and ultracentrifugate at 70000g for 1.5h. Collect the last 1ml medium and resuspend for further experiment.

Selecting stable cell line

1. Seed cells a night before.

2. Refresh medium first.Add polybrene 8ul/ml final concentration. Gently add 100ul virus to every well in a 24-well plate. .

3. Add 1:1000 blastisidin or 1:2000 2mg/ml puromycin to culture medium.

4. Three later, refresh culture medium and passage cells into new plates.

Basic cell culturing

Medium: 10% FBS+ 88% DMEM (high-glucose)+1% P/S (5000 mg/ml penicillin and 5000 mg/ml streptomycin)+1% L-glutamine (200 mM). Cells were passaged with 0.25%Trypsin.

hiPSC culturing

Introduction

Culturing human and pluripotent stem cells requires the use of complex media and careful handling techniques. Here, we describe feeder-free and serum free way with the use of Gibco Essential 8 medium in combination with BD matrigel low growth factor to maintain and propagate high quality hIPSCs.

The following protocol are modified from standard protocols from BD and Gibco

Materials required

Essential 8 medium, consisting of DMEM/F12(AM)1:1 and Essential 8 Supplement(50*)

0.5M EDTA, pH8.0

37°C water bath

BD matrigel low growh factor and Gibco knockout MEM

DPBS without Calcium and Magnesium

Approprate tissue culture plates and supplies

Preparing Media and Materials

Essential 8 Medium (500 mL of complete medium)

1. Thaw Essential 8 Supplement (50X) at 2–8°C overnight instead of at 37°C.

2. Mix the following component into 500 mL of complete Essential 8 Medium s:

DMEM/F-12 (HAM) 1:1 490 mL

Essential 8 Supplement (50X) 10 mL

Note: Before use, warm complete medium at room temperature until it is no longer cool to the touch. Do not warm the medium at 37°C.

0.5 mM EDTA in DPBS (50 mL)

1. To prepare 50 mL of 0.5 mM EDTA in DPBS Mix the following components in a 50-mL conical tube in a biological safety cabinet to prepare 50 mL of 0.5 mM EDTA in DPBS:

DPBS without Calcium and Magnesium 50 mL

0.5 M EDTA 50 µL

2. Filter sterilize the solution. The solution can be stored at room temperature for up to six months.

Coating Culture Vessels with BD matrigel with low growth factor

BD Matrigel low growth factor Matrix should be aliquoted and frozen. Consult the Certificate of Analysis supplied with the BD Matrigel™ for the recommended aliquot size (“Dilution Factor”) to make up 25 mL of diluted matrix. Make sure to always keep BD Matrigel on ice when thawing and handling to prevent it from gelling.

Note: Use tissue culture-treated cultureware (e.g. 6-well plates, BD Catalog #353046).

1. Thaw one aliquot of BD Matrigel on ice.

2. Dispense 25 mL of cold dilution medium (DMEM/F-12; Catalog #36254) into a 50 mL conical tube and keep on ice.

3. Add thawed BD Matrigel to the cold dilution medium (in the 50 mL tube) and mix well. The vial may be washed with cold medium if desired.

4. Immediately use the diluted BD Matrigel solution to coat tissue culture-treated cultureware. for recommended coating volumes.

Passaging iPSCs

In general, split cells when one of the following occurs:

• PSC colonies are becoming too dense or too large.

• PSC colonies are showing increased differentiation.

• The colonies cover approximately 85% of the surface area of the culture vessel, usually every 4 days. Even if the colonies are sparse and small, it is important to split the culture every 4 to 5 days.

The split ratio can vary, though it is generally between 1:2 and 1:4 for early passages and between 1:3 and

1:12 for established cultures. Occasionally, cells will grow at a different rate and the split ratio will need to be adjusted.

• A general rule is to observe the last split ratio and adjust the ratio according to the appearance of the PSC colonies. If the cells look healthy and colonies have enough space, split using the same ratio. If they are overly dense and crowding, increase the ratio. If the cells are sparse, decrease the ratio.

Passaging PSC Colonies using EDTA

Note: Newly derived PSC lines may contain a fair amount of differentiation through passage 4. It is not necessary to remove differentiated material prior to passaging. By propagating/splitting the cells the overall culture health should improve throughout the early passages.

1. Prior to starting, equilibrate your matrigel coated dishes to room temperature in the hood (this takes about one hour). Pre-warm the required volume of Essential 8 Medium at room temperature until it is no longer cool to the touch.

Note: Do not warm medium in a 37°C water bath.

2. Aspirate the spent medium from the vessel containing PSCs with a Pasteur pipette, and rinse the vessel twice with Dulbecco’s PBS (DPBS) without Calcium and Magnesium. Refer to Table 2 for the recommended volumes.

3. Add 0.5 mM EDTA in DPBS to the vessel containing PSCs. Adjust the volume of EDTA for various dish sizes (refer to Table 2). Swirl the dish to coat the entire cell surface.

4. Incubate the vessel at room temperature for 5–8 minutes or 37°C for 4–5 minutes. When the cells start to separate and round up, and the colonies will appear to have holes in them when viewed under a microscope, they are ready to be removed from the vessel.

Note: In larger vessels or with certain cell lines, this may take longer than 5 minutes.

5. Aspirate the EDTA solution with a Pasteur pipette.

6. Add pre-warmed complete Essential 8 Medium to the dish.

7. Remove the cells from the well(s) by gently squirting medium and pipetting the colonies up using a 5-mL glass pipette. Avoid creating bubbles. Collect cells in a 15-mL conical tube.

For 6-well plate, 1ml/well 0.5nM EDTA in DPBS is used to digest cells and 2ml/well complete Essential 8 Medium is used for culturing.

Note: Do not scrape the cells from the dish. There may be obvious patches of cells that were not dislodged and left behind. Do not attempt to recover them through scraping.

Note: Little or no extra pipetting is required to break up cell clumps after EDTA treatment.

Note: Depending upon the cell line, work with no more than one to three wells at a time, and work quickly to remove cells after adding Essential 8 Medium to the well(s). The initial effect of the EDTA will be neutralized quickly by the medium. Some lines re-adhere very rapidly after medium addition, and must be removed 1 well at a time. Others are slower to re-attach, and may be removed 3 wells at a time.

8. Aspirate residual matrigel solution from the pre-coated dish.

9. Add an appropriate volume of pre-warmed Essential 8 Medium to each well of a coated 6-well plate so that each well contains 2 mL medium after the cell suspension has been added. Refer to Table 2 for volumes for other culture vessels.

10. Move the vessel in several quick figure eight motions to disperse cells across the surface of the vessels.

11. Place dish gently into the 37°C, 5% CO 2 incubator and incubate the cells overnight.

12. Feed PSC cells beginning the second day after splitting. Replace spent medium daily.

Note: It is normal to see cell debris and small colonies after passage.