KC-4419

A549-ZRSR2-KO-1A1 Cell Line

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Background of A549-ZRSR2-KO-1A1 Cell Line

ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) is an essential splicing factor involved in the recognition of 3′ splice sites during pre-mRNA splicing. Pre-mRNA splicing is carried out by the major and minor spliceosomes, which target U2- and U12-type introns, respectively. Mutations in ZRSR2 have been associated with various diseases, including X-linked intellectual disability and cancer. The gene is located on the X chromosome and is highly conserved across species.

Specifications

Catalog Number	KC-4419
Cell Line Name	A549-ZRSR2-KO-1A1 Cell Line
Host Cell Line	A549
Description	Stable A549 clone with human ZRSR2 gene knockout, No.1A1
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	McCoy's 5A+20% FBS+10% DMSO
Propagation Medium	McCoy's 5A+10%FBS+L-Glutamax
Selection Marker	NA
Morphology	epithelial
Subculture	Split saturated culture 1:4-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

A549-ZRSR2-KO-1A1 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of A549-ZRSR2-KO-1A1 cell line stable clone using PCR sequencing.

Figure 2: Characterization of A549-ZRSR2-KO-1A1 cell line stable clone using RT-PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (McCoy's 5A+10%FBS+L-Glutamax)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:4-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% McCoy's 5A + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage

References

1.Weinstein R, Bishop K, Broadbridge E, Yu K, Carrington B, Elkahloun A, Zhen T, Pei W, Burgess SM, Liu P, Bresciani E, Sood R. Zrsr2 Is Essential for the Embryonic Development and Splicing of Minor Introns in RNA and Protein Processing Genes in Zebrafish. Int J Mol Sci. 2022 Sep 14;23(18):10668. doi: 10.3390/ijms231810668. PMID: 36142581; PMCID: PMC9501576.