BioVector® CH12F3 (APE1-/-/- APE2-) 基因敲除小鼠B淋巴瘤细胞株

BioVector® CH12F3 (APE1-/-/- APE2-) Triple/Quadruple Knockout Mouse B-Lymphoma Cell Line

第一部分 中文说明

一 产品基本信息与遗传学背景

• 细胞名称：BioVector® CH12F3 ($APE1^{-/-/-}\ APE2^{-}$) 小鼠B淋巴细胞瘤双基因完全敲除细胞

• 系统学 Accession：Cellosaurus CVCL_GZ22

• 物种来源：小鼠 (Mus musculus)

• 组织与疾病背景：源自经典的小鼠B细胞淋巴瘤株 CH12F3（其亲本株为 CH12.LX）。CH12F3 是一类在体外经细胞因子（如 IL-4、TGF-beta 和 anti-CD40）联合诱导下，能够发生超高效率免疫球蛋白重链类别转换重组（Class Switch Recombination, CSR）的标志性模型。

• 双基因缺陷特征 ($APE1^{-/-/-}\ APE2^{-}$)：

  • 三拷贝 APE1 完全敲除 ($APE1^{-/-/-}$)：CH12F3 属于近三倍体（Hypotriploid）细胞。该细胞通过染色体常规同源重组技术，将其基因组中全部 3个拷贝 的内源性脱嘌呤/脱嘧啶核酸内切酶1基因（Apex1 基因，编码 APE1）彻底删除。由于 APE1 基因在普通小鼠胚胎和干细胞中敲除会导致早期致死，该株是极少数成功创制的哺乳动物 APE1 零表达株。

  • APE2 协同敲除 ($APE2^{-}$)：在 APE1 缺失的背景下，进一步将次要的 AP 核酸内切酶2基因（Apex2 基因，编码 APE2）靶向敲除，从而在细胞内彻底阻断了整个碱基切除修复（BER）途径中的无碱基（AP）位点单链切开通路。

• 生物安全级别：1级（BSL-1）。

二 细胞形态学与培养环境

• 形态学特征：展现典型的B淋巴母细胞样、圆形的单细胞形态。细胞在液体中呈不贴壁的分散悬浮状态生长，有时会呈现极轻微的小聚集簇或双细胞对，胞质内带有丰富的免疫球蛋白代谢相关超微结构。

• 生长模式：悬浮生长。

• 倍增时间：增殖动力学相对活跃，倍增时间大约为 20 到 26 小时。令人惊讶的是，尽管完全缺乏核心的 APE1/APE2 DNA修复酶，该细胞在常规无压力培养下仍维持了与野生型（WT）高度类似的细胞存活率与生长曲线。

• 标准完全培养基配方：

  • 基础培养基：BioVector® RPMI-1640 培养基。

  • 维持添加：

    • 10% 到 15% BioVector® 优质胎牛血清（较高血清配比利于保护 DNA 修复缺陷株的复苏早期活力）。

    • 50 μM 2-巯基乙醇（2-Mercaptoethanol，2-ME，必需添加项，维持B细胞特异性氧化还原状态）。

• 物理培养参数：37摄氏度恒温、5% 二氧化碳、空气饱和湿度。

三 细胞传代与复苏标准操作步骤

1. 常规传代操作：

   • 当悬浮细胞密度达到每毫升 $8.0 \times 10^5$ 至 $1.5 \times 10^6$ 个活细胞，或者培养基由于乳酸蓄积由红变黄时，必须进行传代。

   • 将细胞悬液转移至无菌离心管中，以 150 g 离心 5 分钟，彻底弃去旧培养基。

   • 加入新鲜预热的 BioVector® 含有 2-巯基乙醇的完全培养基重悬。

   • 推荐起始接种密度：控制在每毫升 $1.5 \times 10^5$ 至 $2.0 \times 10^5$ 个活细胞。常规传代比例为 1比3 至 1比5，每周需要补充或更换培养基 2 到 3 次。由于该株缺乏主要 AP 内切酶，培养过程中切勿使其暴露于阳光暴晒或过高强度的过氧化物环境中。

2. 冻存细胞复苏：

   • 从液氮中取出冷冻管，立即投入 37摄氏度 BioVector® 水浴锅中高频摇动使其融化，严格控制在 2 分钟以内。

   • 迅速移入 5 mL 预热基础培养基试管，150 g 离心 5 分钟以去除残留的 DMSO。

   • 弃上清，加入新鲜完全培养基重悬，接种后正常静置培养。

四 核心科研应用方向

1. 免疫球蛋白类别转换重组（CSR）分子机制研究：BioVector® CH12F3 ($APE1^{-/-/-}\ APE2^{-}$) 是全球免疫学和 DNA 损伤修复领域极为珍贵的金标准工具细胞。在此细胞中，由诱导性胞苷脱氨酶（AID）触发、UNG2 糖苷酶切除产生的 DNA 环状无碱基（AP）位点将无法再被 APE1/APE2 正常切开，导致其从 IgM 向 IgA 的类别转换效率发生戏剧性的暴跌（仅剩野生型的约20%或更低）。常用于解析在缺乏 BER 经典酶的情况下，MRE11/CtIP 或错配修复（MMR）系统对 S区 双链断裂（DSBs）的替代性剪切机制。

2. AP 碱基切除修复（BER）底物与脱靶效应评估：由于同时缺乏 APE1 和 APE2 活性，该细胞是鉴定新型小分子 APE1 内切酶抑制剂（如 Compound 3）和氧化还原抑制剂（如 APX2009）是否存在细胞内脱靶效应（Off-target effects）的绝对对照模型（若靶向特异，敲除株应对抑制剂表现出天然耐药或不敏感）。

3. 烷化剂诱导突变谱与遗传毒理学测试：由于其完全丧失了 AP 位点的经典剪切修复能力，该细胞对甲磺酸甲酯（MMS）、替莫唑胺等 DNA 烷化剂类药物展现出极端的超敏感性（Hypersensitivity）。常用于高通量测定环境化学毒物的单链断裂转化率、诱导染色体互换以及碱基错配发生动力学。

PART 2 ENGLISH SECTION

I General Information and Genetic Background

• Cell Line Name: BioVector® CH12F3 ($APE1^{-/-/-}\ APE2^{-}$)

• Synonyms: CH12F3-APE1/APE2-KO, CH12F3 (APE1-/-/-APE2-)

• Cellosaurus Accession: CVCL_GZ22

• Species Origin: Mouse (Mus musculus)

• Tissue and Disease Background: Derived from the established murine B-cell lymphoma line CH12F3 (originally from parent clone CH12.LX). CH12F3 represents a premier textbook mammalian system capable of executing high-efficiency Immunoglobulin heavy chain Class Switch Recombination (CSR) from IgM to IgA upon synchronized cytokine cocktail induction (IL-4, TGF-beta, and anti-CD40 antibodies) in vitro.

• Genomic Knockout Architecture ($APE1^{-/-/-}\ APE2^{-}$):

• • Triple-Allele APE1 Deletion ($APE1^{-/-/-}$): Since the hypotriploid CH12F3 line harbors 3 functional genomic copies of the apurinic/apyrimidinic endonuclease 1 (Apex1) gene, homologous recombination was deployed to completely eliminate all 3 alleles. While APE1 ablation is notoriously embryonic lethal in normal mouse tissues and embryonic stem cells, this specialized background represents a viable, stable 0-expression null clone.

  • Concomitant APE2 Ablation ($APE2^{-}$): To completely plug overlapping backup nuclease networks processing abasic damage, the secondary AP endonuclease 2 gene (Apex2) was targeted and disrupted, inactivating the core backbone of the Base Excision Repair (BER) pathway at apurinic/apyrimidinic interfaces.

• Biosafety Level: BSL-1.

II Morphological Attributes and Cultivation Media

• Morphology Displays uniform lymphoblastoid, round single-cell contours. Cells proliferate strictly in a non-adherent, single-cell suspension layout, occasionally gathering into tiny doublets or micro-pairs, preserving full B-lineage hyper-metabolic organelles.

• Growth Mode Suspension growth.

• Cell Doubling Interval Proliferates dynamically with a doubling period of approximately 20 to 26 hours. Surprisingly, despite total deprivation of its primary AP endonucleases, the line maintains excellent baseline survival profiles and cell cycle curves similar to wild-type equivalents under routine expansion.

• Standard Complete Growth Medium Formulation

  • Basal Medium: BioVector® RPMI-1640 medium.

  • Routine Supplements:

    • 10% to 15% premium BioVector® Fetal Bovine Serum (FBS). Higher serum parameters protect emerging double-knockout clones post-thaw.

    • 50 μM 2-Mercaptoethanol (2-ME, mandatory constituent to preserve mandatory intracellular redox baselines for B-lymphocytes).

• Physical Incubation Thresholds Regulated strictly at 37 degrees Celsius under an atmospheric layer of 5% Carbon Dioxide and saturated air humidity.

III Subculturing and Thawing Protocols

1. Routine Passaging Schedule:

   • Subculture the biomass when the active suspension density bridges $8.0 \times 10^5$ to $1.5 \times 10^6$ viable cells/mL, or when the phenol red parameter indicates substantial lactic acid buildup.

   • Spin the entire suspension slurry at 150 g for 5 minutes, completely draw off the spent broth, and resuspend the pellet.

   • Recommended Seeding Thresholds: Establish fresh seeding densities at $1.5 \times 10^5$ to $2.0 \times 10^5$ viable cells/mL. The standard split sequence ranges from 1比3 to 1比5, demanding medium replenishment or passaging 2 to 3 times per week. Avoid exposing this DNA-repair deficient line to solar irradiation or high-intensity hydrogen peroxide stresses.

2. Cryovial Thawing and Recovery:

   • Retrieve the cryovial from liquid nitrogen and submerge it into a 37 degrees Celsius BioVector® water bath with rapid agitation until completely liquefied within 2 minutes.

   • Promptly dilute the mixture into a centrifuge tube containing 5 mL of pre-warmed complete growth medium and pellet at 150 g for 5 minutes to eliminate DMSO traces.

   • Decant the supernatant, gently resuspend the cell pellet in fresh, 2-ME enriched complete BioVector® medium, and plate.

IV Strategic Research Applications

1. Dissecting Molecular Cascades of Class Switch Recombination (CSR): BioVector® CH12F3 ($APE1^{-/-/-}\ APE2^{-}$) represents a rare genetic tool in molecular immunology and genomic stability labs. In this line, the genomic abasic (AP) targets generated by AID/UNG2 within switch regions cannot be cleared by APE1/APE2, causing standard IgM-to-IgA class switching efficiency to plummet (~20% or less of wild-type benchmarks). It is extensively used to trace alternative switch cleavage circuits led by the MRE11/CtIP complex or components of the Mismatch Repair (MMR) apparatus.

2. Validating BER Targeted Inhibitors & Off-Target Actions: Lacking all endogenous APE1/APE2 cleavage actions, this system provides an absolute negative control model to identify whether new small-molecule APE1 endonuclease blockers (e.g., Compound 3) or redox axis modulators (e.g., APX2009) present cellular off-target toxicities (a truly specific APE1 inhibitor should exhibit zero added cytostatic effects on an already APE1-null baseline).

3. Alkylating Agent Genotoxicity and Mutational Fingerprinting: Devoid of early AP-site strand incision capabilities, this double-knockout model displays severe hypersensitivity to clinical alkylating agents like Methyl Methanesulfonate (MMS) or Temozolomide. It serves as a standard assay platform to profile single-strand break accumulation rates, replication fork collapse, and translesion synthesis kinetics under environmental genotoxic stress.

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