Confocal Fluorescence Microscopy of CRISPR-generated G6PD Knockout mouse liver cells (TIB-73)

IN Cell Culture, Discussion Topics, Featured Research

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas-9 knockout of glucose 6-phosphate dehydrogenase (G6PD)



The first surviving G6PD- knockout somatic cell line was generated with CRISPR-Cas-9 technology to support subcellular localization studies. G6PD,  glucose 6-phosphate dehydrogenase, is the rate-limiting enzyme in the pentose phosphate pathway. G6PD is involved in oxidative stress, apoptosis, angiogenesis and cancer proliferation among other functions (2). In fact, elevated levels of expression and activity of G6PD are frequently observed in breast, colon, endometrial, cervical, prostatic, and lung cancer and G6PD is considered to be a potential therapeutic target. G6PD is also essential for life. Early studies show embryonic lethality of rodent models, indicating that viable G6PD knockout mice cannot be produced.  It was postulated that somatic cells cannot survive knockout of G6PD.  However, this study demonstrates that the mouse hepatic cell line TIB-73 can survive G6PD knockout (1) and that CS16-CultureWell™ Removable Chambered Coverglass (Item # 112358) is the ideally suited to for live-cell imaging experiments in subcellular localization studies.

Cloning and Transfection

TIB-73 cells, an immortalized strongly adherent mouse liver cell line, were grown in 6-well tissue culture plates.  Cloning and transfection of Cas9 and mouse G6PD-targeted sgRNA plasmids (4) were performed as described in Spencer et. al (1). A pTRE2-pur plasmid (3) was co-transfected to confer puromycin resistance to transfected cells.  Puromycin treatment was discontinued 24 hours post-transfection to ensure that the cells would not begin to integrate pTRE2-pur into their genomic DNA.  After puromycin treatment, about 20% of the cells remained alive.  Transfected cells were trypsinized and plated into 96-well plates at a limiting dilution density calculated to be 1 cell per well.  Cells that grew as clonal populations were expanded over time to grow in 6-well plates. These large clonal populations of CRISPR-treated cells were rapidly screened for G6PD activity (5).  G6PD knockout cell populations were identified based on DNA-sequencing and confirmed by Western Blotting.

G6PD subcellular localization studies via live-cell imaging 

Live cell imaging of G6PD knockout cells was utilized to study the subcellular localization of G6PD and to identify the nuclear localization signal. These studies are excellent to study monomeric proteins but for multimeric proteins, the interaction of the native protein with fluorescently tagged-mutants can cause artifacts. Because G6DP is capable of homodimerization G6PD know-out cells were used in this experiment to rule out this interference.  Thus, G6PD knockout cells (Tib73) were propagated on CS16-CultureWell™ Removable Chambered Coverglass (Item # 112358)
and transfected with various plasmid constructs to express full-length G6PD-mCherry, N-terminal-G6PD-mCherry, and C-terminal-G6PD-mCherry mutants. Transfected Tib73 cells were then imaged live by laser scanning confocal microscopy.

Experimental flowchart

Conclusions

The first G6PD-knocked somatic cell line ever produced was used to overcome experimental limitations of subcellular localization studies.  CS16-CultureWell™ Removable Chambered Coverglass is a cost-effective and time-saving solution for this type of experiment. G6PD knockout cells can be cultured directly on the untreated glass bottom of the chamber and transfected with a small volume of reagent.  Fixing and staining, as well as live-cell imaging, can all be performed in the in the same chamber. The G6PD knockout cell line is a convenient tool for studying G6PD role in the survival, proliferation, and metastasis of cancer cells as well as in the development of a potential novel class of inhibitors for cancer therapy.

References

1. Spencer, N. Y., Yan, Z., Cong, L., Zhang, Y., Engelhardt, J. F., and Stanton, R. C. (2016) Definitive localization of intracellular proteins: Novel approach using CRISPR-Cas9 genome editing, with glucose 6-phosphate dehydrogenase as a model, Analytical Biochemistry 494, 55-67.

2. Spencer, N.Y., and Stanton, R.C. (2017) Glucose 6-Phosphate Dehydrogenase and the Kidney, Current Opinion in Nephrology and Hypertension, 26, 43-49.

3. Clontech

4. Lipofectamine LTX with Plus Reagent.

5. Life Technologies Vybrant Cytotoxicity Assay Kit (V-23111).

Cells

G6PD Knockout Cells:

https://www.atcc.org/en/Products/Cells_and_Microorganisms/By_Tissue/Liver/CRL-3309.aspx

G6PD Wild-type Control (“high-passage”) Cells:

https://www.atcc.org/en/Products/Cells_and_Microorganisms/By_Tissue/Liver/CRL-3308.aspx