Protein electroporation of Cre recombinase into cultured Arabidopsis cells with an intact cell wall CURRENT

Cre recombinase is widely used in a variety of organisms to manipulate DNA both in vitro and in vivo. Combined with its consensus DNA sequences, named loxP, it allows the DNA modification including deletions, insertions, translocations and inversions at specific DNA locus of cells. To promote its use in plant cultured cells, we report here a detailed protocol for direct protein delivery of Cre recombinase into cultured Arabidopsis thaliana cells with an intact cell wall. Electroporation with the optimized buffer and program enables the efficient delivery of Cre protein and following recombination in cultured Arabidopsis thaliana cells. Specifically, we describe the purification of Cre protein, the preparation of electroporation samples, and the assessment of electroporated cells. This simple, economical, and effective technology will contribute to the biological analysis of genes and cellular functions in cultured Arabidopsis thaliana cells. This protocol accompanies Furuhata et al (DOI: 10.1038/s41598-018-38119-9), Scientific Reports , published online on February 15th 2019.


Introduction
The Cre/loxP system is a powerful genome engineering tool that has been extensively used in numerous areas of the life sciences. Cre recombinase catalyzes recombination between two of its consensus DNA sequences, named loxP 1-3 , and can be used for various applications including lineage/cell type-specific gene knockout, selectable marker removal, and chromosomal rearrangement [1][2][3][4][5][6][7][8] . Although the Cre/loxP system has been widely used in a variety of organisms including plant cells [9][10][11][12][13][14][15][16][17] , its application in cultured plant cells has been limited compared to that in animal cells. One major obstacle is cell wall, which protects plant cells from mechanical stress and the entry of exogenous substances. Methods have been developed to deliver biomaterials into cultured plant cells through the cell wall, such as the delivery of DNA using a particle gun or Agrobacterium (Rhizobium)-mediated techniques are widely used in plant research 18,19 . However, exogenous DNA fragments are often or invariably incorporated into the genomic DNA and may induce unexpected effects that interfere with subsequent analyses. To overcome these obstacles, we focus on direct protein delivery to cultured plant cells and show an efficient protein-electroporation technique for Cre recombinase into cultured Arabidopsis thaliana cells with an intact cell wall.
3) Add 100 μL of SOC media to the mixture and incubate at 37°C for 30-60 min. 2) Incubate at 37°C with shaking for 2-3 h, until absorbance A 600 becomes 0.6.
3) Shift the temperature to 18°C and incubate with shaking for 30 min.  3) Add 800 μl of 10% Triton X-100 towards final concentration of 0.2% Triton X-100. 4) Sonicate the suspension to shear the DNA for 10 min on ice. We do not add lysozyme or nuclease to the lysate, since these components are difficult to be removed completely from samples.

5)
Centrifuge the suspension at 9,000 g for 10 min at 4°C to pellet the cellular debris. 2) Freeze the purified HNCre protein using liquid N 2 and store it at -80°C 3) Thaw the frozen protein and dialyze it with HBS before use.
Note: It is important to use buffers containing 500 mM NaCl because Cre protein, especially at lower salt concentration, tends to bind and thus copurified with nucleic acids, and also tends to aggregate during purification process. Note: When T87-xGxGUS cells is used, the delivery efficiency of HNCre protein can be measured by ßglucuronidase (GUS) activity using X-Glucuronide or 6-chloro-4-methylumbelliferyl β-D-glucuronide, or genomic PCR primers flanking the two loxP sites as follows.

GUS staining
1) Two days after electroporation, discard the supernatant and resuspend cells with 0.5 mg/mL of X-Glucuronide dissolved in 300 μL of staining buffer.
Note: At least 2 days of incubation after electroporation are necessary for the detection of GUS activity.

Fluorescence quantification of GUS activity
1) Two days after electroporation, wash cells once with NT1 medium (pH 7.0).
2) Suspend cells with 5 µL of PCV in 100 µL of NT1 medium (pH 7.0).    The 1307 bp and 151 bp fragments represent the reporter gene cassette before and after

3) Transfer cells to a black
Cre-mediated recombination, respectively. In all assays, cells were electroporated in Opti-MEM I containing 5 μM Cre protein using 5 poring pulses of 150 V and 10 ms. Assays were performed at 2 days after electroporation. Control indicates untreated T87-xGxGUS cells.

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A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall by Yuichi Furuhata, Ayako Sakai, Tomi Murakami, +6 Scientific Reports (11 March, 2019)