BioVector? CH12F3 (APE1-/-/- APE2-) 基因敲除小鼠B淋巴瘤細(xì)胞株

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

第一部分 中文說(shuō)明

一 產(chǎn)品基本信息與遺傳學(xué)背景

• 細(xì)胞名稱：BioVector? CH12F3 ($APE1^{-/-/-}\ APE2^{-}$) 小鼠B淋巴細(xì)胞瘤雙基因完全敲除細(xì)胞

• 系統(tǒng)學(xué) Accession：Cellosaurus CVCL_GZ22

• 物種來(lái)源：小鼠 (Mus musculus)

• 組織與疾病背景：源自經(jīng)典的小鼠B細(xì)胞淋巴瘤株 CH12F3（其親本株為 CH12.LX）。CH12F3 是一類在體外經(jīng)細(xì)胞因子（如 IL-4、TGF-beta 和 anti-CD40）聯(lián)合誘導(dǎo)下，能夠發(fā)生超高效率免疫球蛋白重鏈類別轉(zhuǎn)換重組（Class Switch Recombination, CSR）的標(biāo)志性模型。

• 雙基因缺陷特征 ($APE1^{-/-/-}\ APE2^{-}$)：

  • 三拷貝 APE1 完全敲除 ($APE1^{-/-/-}$)：CH12F3 屬于近三倍體（Hypotriploid）細(xì)胞。該細(xì)胞通過(guò)染色體常規(guī)同源重組技術(shù)，將其基因組中全部 3個(gè)拷貝 的內(nèi)源性脫嘌呤/脫嘧啶核酸內(nèi)切酶1基因（Apex1 基因，編碼 APE1）徹底刪除。由于 APE1 基因在普通小鼠胚胎和干細(xì)胞中敲除會(huì)導(dǎo)致早期致死，該株是極少數(shù)成功創(chuàng)制的哺乳動(dòng)物 APE1 零表達(dá)株。

  • APE2 協(xié)同敲除 ($APE2^{-}$)：在 APE1 缺失的背景下，進(jìn)一步將次要的 AP 核酸內(nèi)切酶2基因（Apex2 基因，編碼 APE2）靶向敲除，從而在細(xì)胞內(nèi)徹底阻斷了整個(gè)堿基切除修復(fù)（BER）途徑中的無(wú)堿基（AP）位點(diǎn)單鏈切開(kāi)通路。

• 生物安全級(jí)別：1級(jí)（BSL-1）。

二 細(xì)胞形態(tài)學(xué)與培養(yǎng)環(huán)境

• 形態(tài)學(xué)特征：展現(xiàn)典型的B淋巴母細(xì)胞樣、圓形的單細(xì)胞形態(tài)。細(xì)胞在液體中呈不貼壁的分散懸浮狀態(tài)生長(zhǎng)，有時(shí)會(huì)呈現(xiàn)極輕微的小聚集簇或雙細(xì)胞對(duì)，胞質(zhì)內(nèi)帶有豐富的免疫球蛋白代謝相關(guān)超微結(jié)構(gòu)。

• 生長(zhǎng)模式：懸浮生長(zhǎng)。

• 倍增時(shí)間：增殖動(dòng)力學(xué)相對(duì)活躍，倍增時(shí)間大約為 20 到 26 小時(shí)。令人驚訝的是，盡管完全缺乏核心的 APE1/APE2 DNA修復(fù)酶，該細(xì)胞在常規(guī)無(wú)壓力培養(yǎng)下仍維持了與野生型（WT）高度類似的細(xì)胞存活率與生長(zhǎng)曲線。

• 標(biāo)準(zhǔn)完全培養(yǎng)基配方：

  • 基礎(chǔ)培養(yǎng)基：BioVector? RPMI-1640 培養(yǎng)基。

  • 維持添加：

    • 10% 到 15% BioVector? 優(yōu)質(zhì)胎牛血清（較高血清配比利于保護(hù) DNA 修復(fù)缺陷株的復(fù)蘇早期活力）。

    • 50 μM 2-巰基乙醇（2-Mercaptoethanol，2-ME，必需添加項(xiàng)，維持B細(xì)胞特異性氧化還原狀態(tài)）。

• 物理培養(yǎng)參數(shù)：37攝氏度恒溫、5% 二氧化碳、空氣飽和濕度。

三 細(xì)胞傳代與復(fù)蘇標(biāo)準(zhǔn)操作步驟

1. 常規(guī)傳代操作：

   • 當(dāng)懸浮細(xì)胞密度達(dá)到每毫升 $8.0 \times 10^5$ 至 $1.5 \times 10^6$ 個(gè)活細(xì)胞，或者培養(yǎng)基由于乳酸蓄積由紅變黃時(shí)，必須進(jìn)行傳代。

   • 將細(xì)胞懸液轉(zhuǎn)移至無(wú)菌離心管中，以 150 g 離心 5 分鐘，徹底棄去舊培養(yǎng)基。

   • 加入新鮮預(yù)熱的 BioVector? 含有 2-巰基乙醇的完全培養(yǎng)基重懸。

   • 推薦起始接種密度：控制在每毫升 $1.5 \times 10^5$ 至 $2.0 \times 10^5$ 個(gè)活細(xì)胞。常規(guī)傳代比例為 1比3 至 1比5，每周需要補(bǔ)充或更換培養(yǎng)基 2 到 3 次。由于該株缺乏主要 AP 內(nèi)切酶，培養(yǎng)過(guò)程中切勿使其暴露于陽(yáng)光暴曬或過(guò)高強(qiáng)度的過(guò)氧化物環(huán)境中。

2. 凍存細(xì)胞復(fù)蘇：

   • 從液氮中取出冷凍管，立即投入 37攝氏度 BioVector? 水浴鍋中高頻搖動(dòng)使其融化，嚴(yán)格控制在 2 分鐘以內(nèi)。

   • 迅速移入 5 mL 預(yù)熱基礎(chǔ)培養(yǎng)基試管，150 g 離心 5 分鐘以去除殘留的 DMSO。

   • 棄上清，加入新鮮完全培養(yǎng)基重懸，接種后正常靜置培養(yǎng)。

四 核心科研應(yīng)用方向

1. 免疫球蛋白類別轉(zhuǎn)換重組（CSR）分子機(jī)制研究：BioVector? CH12F3 ($APE1^{-/-/-}\ APE2^{-}$) 是全球免疫學(xué)和 DNA 損傷修復(fù)領(lǐng)域極為珍貴的金標(biāo)準(zhǔn)工具細(xì)胞。在此細(xì)胞中，由誘導(dǎo)性胞苷脫氨酶（AID）觸發(fā)、UNG2 糖苷酶切除產(chǎn)生的 DNA 環(huán)狀無(wú)堿基（AP）位點(diǎn)將無(wú)法再被 APE1/APE2 正常切開(kāi)，導(dǎo)致其從 IgM 向 IgA 的類別轉(zhuǎn)換效率發(fā)生戲劇性的暴跌（僅剩野生型的約20%或更低）。常用于解析在缺乏 BER 經(jīng)典酶的情況下，MRE11/CtIP 或錯(cuò)配修復(fù)（MMR）系統(tǒng)對(duì) S區(qū) 雙鏈斷裂（DSBs）的替代性剪切機(jī)制。

2. AP 堿基切除修復(fù)（BER）底物與脫靶效應(yīng)評(píng)估：由于同時(shí)缺乏 APE1 和 APE2 活性，該細(xì)胞是鑒定新型小分子 APE1 內(nèi)切酶抑制劑（如 Compound 3）和氧化還原抑制劑（如 APX2009）是否存在細(xì)胞內(nèi)脫靶效應(yīng)（Off-target effects）的絕對(duì)對(duì)照模型（若靶向特異，敲除株應(yīng)對(duì)抑制劑表現(xiàn)出天然耐藥或不敏感）。

3. 烷化劑誘導(dǎo)突變譜與遺傳毒理學(xué)測(cè)試：由于其完全喪失了 AP 位點(diǎn)的經(jīng)典剪切修復(fù)能力，該細(xì)胞對(duì)甲磺酸甲酯（MMS）、替莫唑胺等 DNA 烷化劑類藥物展現(xiàn)出極端的超敏感性（Hypersensitivity）。常用于高通量測(cè)定環(huán)境化學(xué)毒物的單鏈斷裂轉(zhuǎn)化率、誘導(dǎo)染色體互換以及堿基錯(cuò)配發(fā)生動(dòng)力學(xué)。

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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