KC-5742

293T-mem-KLK2-D207N Cell Line

59775

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Background of 293T-mem-KLK2-D207N Cell Line

Human kallikrein-related peptidase 2 (KLK2) is a tryptic serine protease predominantly expressed in prostatic tissue and secreted into prostatic fluid, a major component of seminal fluid, it is a Protein Coding gene. This gene encodes a member of the grandular kallikrein protein family. Kallikreins are a subgroup of serine proteases that are clustered on chromosome 19. Members of this family are involved in a diverse array of biological functions. The protein encoded by this gene is a highly active trypsin-like serine protease that selectively cleaves at arginine residues. This protein is primarily expressed in prostatic tissue and is responsible for cleaving pro-prostate-specific antigen into its enzymatically active form. Diseases associated with KLK2 include Prostate Disease and Prostate Cancer.

Specifications

Catalog Number	KC-5742
Cell Line Name	293T-mem-KLK2-D207N Cell Line
NCBI/UniProt Accession Number	NP_005542.1
Clone Number	3#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous mem KLK2 bearing D207N spontaneous mutation
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	70% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 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

293T-mem-KLK2-D207N cell line was generated using a lentiviral vector expressing the mem KLK2 D207N sequence.

Characterization

Figure 1: Characterization of mem KLK2 overexpression in the 293T-mem-KLK2 stable clone using FACS.

Figure 2: Characterization of mem KLK2 in the 293T stable clone using PCR sequencing.(Spontaneous mutation: D207N)

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 1μg/mL Puromycin)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 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 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. Lövgren J, Valtonen-André C, Marsal K, Lilja H, Lundwall A. Measurement of prostate-specific antigen and human glandular kallikrein 2 in different body fluids. J Androl. 1999 May-Jun;20(3):348-55. PMID: 10386814.
2. Batra J, O'Mara T, Patnala R, Lose F, Clements JA. Genetic polymorphisms in the human tissue kallikrein (KLK) locus and their implication in various malignant and non-malignant diseases. Biol Chem. 2012 Dec;393(12):1365-90. doi: 10.1515/hsz-2012-0211. PMID: 23667899.
3.Skala W, Utzschneider DT, Magdolen V, Debela M, Guo S, Craik CS, Brandstetter H, Goettig P. Structure-function analyses of human kallikrein-related peptidase 2 establish the 99-loop as master regulator of activity. J Biol Chem. 2014 Dec 5;289(49):34267-83. doi: 10.1074/jbc.M114.598201. Epub 2014 Oct 16. PMID: 25326387; PMCID: PMC4256358.