Procotol of VIKING method

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The protocol for using HaCaT cells or HEK293F cells is shown.

◆Materials

· Target genome cleavage vector (we use pX330 https://www.addgene.org/42230/)

· Donor vector* (pVKG1_PURO, puromycin resistance, planned to be distributed from Addgene)

· Donor cleavage vector* (pX330_VKG1, planned to be distributed from Addgene)

*The donor cutting vector and the donor vector are known as the ‘VIKING module’. This is available independently of the target genome sequence.

It is scheduled to be distributed via Addgene, but for immediate request please contact shun-sawa2 @ tokushima-u.ac.jp or sugano @ fc.ritsumei.ac.jp.

◆Experimental procedure

Step 1: Cell culture

HaCaT cells or human embryonic kidney cells 293F (HEK293F) cells are incubated in D10 medium comprising Dulbecco’s modified Eagle’s medium (DMEM) (043-30085; Wako, Osaka, Japan) with 10% fetal calf serum (S1780-500; Biowest, Nuaille, France) supplemented with 1 U penicillin–streptomycin (161-23181; Wako).

Step 2: Transformation

-Electroporation method

HaCaT cells are suspended in Opti-MEM (11058-021; Life Technologies), and the VIKING module and pX330 plasmids totaling 15 µg DNA (molar ratio donor cleavage plasmid (pX330_VKG1): donor plasmid: target cleavage plasmid = 1: 1: 17) are transfected by electroporation using an electroporator (CUY21 EDITII; Bex, Tokyo, Japan). Electroporated cells are transferred to 100 mm dishes and precultured for 24 h in D10 medium without antibiotics.

-Lipofection method

The VIKING module and pX330 plasmids totaling 15 µg (molar ratio, PX330_VKG1: donor plasmid: target cut plasmid = 1: 1: 17) DNA are transfected into HEK293F cells using TurboFect transfection reagent (R0531; Thermo Fisher Scientific, Waltham, MA). Transfected cells are transferred to a 100 mm dish and precultured for 24 h in D10 medium without antibiotics.

Step 3: Selection

Transformed cells are developed in culture medium. Puromycin (0.3 μg/mL) is added to D10 medium and cells are cultured for 14 days.

A single colony showing resistance to puromycin is isolated and transferred to a 24-well plate.

After culturing for several days, cells are transferred to a 100 mm dish and incubated.

Step 4: DNA extraction

DNA extraction is performed from some of the selected cells for genotyping. DNA extraction follows conventional protocols, such as

https://openwetware.org/wiki/DNA_extraction_from_tissue

Step 5: Genotyping (confirmation of knock-in by PCR)

Using the extracted DNA as a template, PCR with GoTaq Polymerase (Promega, M3001) is used to confirm knock-in insertion and random insertion.

There is no need to use special enzymes for PCR.

◆Required primer design

Figure: Schematic of primers for genotyping the knock-in

-Design primers to amplify the VKG1 sequence (Fig.).

Example:

Primer 1 (VKG 1 _ 4561 F), 5'-GCCTATGGAAAAACGCCAGC-3'

Primer 2 (VKG 1 _ 250 R), 5'-TTCCTGTCTAGCGGTACGCG-3'

-Design primers that specifically bind to the target genome sequence.

Example:

Primer 3 (VDR genome _ del 3 F), 5'-GGTGGGCCTCATGTCTTCTG-3'

Primer 4 (VDR genome_del 1 R), 5'-CCTTCATCATGCCGATGTCC-3'

◆Genotyping by PCR

PCR reaction

2´ GoTaq® GreenMaster Mix 12.5 µL

10 µM Primer A 2.5 µL

10 µM Primer B 2.5 µL

Nuclease-free water 6.5 µL

Genomic DNA 1 µL

Total 25 µL

Thermocycler cycle

95° C 2 min

[95° C for 30 s, 55°C for 1 min, 72°C for 2 min] ´ 30 cycles

72°C 4 min

12°C hold

Step 1. Confirm random insertion

PCR is performed using Primer 1 and Primer 2.

If DNA fragment 1 is amplified, random insertion is confirmed.

Step 2. Confirm knock-in to the target genome

- Forward

PCR is performed using Primer 1 and Primer 3, or Primer 2 and Primer 4.

If DNA fragment 2 and fragment 3 are amplified, forward knock-in is confirmed.

-Reverse direction

PCR is performed using Primer 2 and Primer 3, or Primer 1 and Primer 4.

If DNA fragment 4 and fragment 5 are amplified, it knock-in in the opposite direction is confirmed.