基因轉殖小鼠生產暨十字形 DNA 結構與基因重組之關聯性研究

Abstract

醫藥用蛋白質量產方法的開發是生物相似藥生產的重要項目之一。哺乳類動物的乳腺是一種高產量且轉譯後修飾與人體相似的生物反應器。本研究室於克弗爾發酵乳中分離出抗高血壓胜肽 (Anti-hypertension Peptide; AP1)。本研究欲使用不同轉殖技術製造乳腺專一性表現 AP1 之基因轉殖小鼠。結果顯示直接施打線性 DNA (8.7%) 產製基因轉殖小鼠效率較使用 CRISPR/Cas9 技術 (5.6%) 效率接近。胚胎移殖存活率於施打線性 DNA 組 (39%) 較使用 CRISPR/Cas9 技術 (16%) 之存活率稍高。本研究成功製備三品系乳腺專一性表現抗高血壓胜肽AP1之基因轉殖小鼠，分別為： FVB-Tg(αLA-AP1x1) 、 FVB-Tg(αLA-AP1x6)4 及 ICR-Gt(ROSA)26Sorem(αLA-AP1x6)BM2 。欲比較三種不同數量 AP1 的 CDS 序列及不同轉殖法生產之小鼠，其乳腺 AP1 胜肽的產能差異。然而，本研究中顯示此三品系基因轉殖小鼠在乳腺中 AP1 mRNA 產量皆低落。並且，僅成功經由 HPLC 及 LC/MS 辨識出 FVB-Tg(αLA-Ap1x1) 鼠乳含有 AP1 胜肽。而三品系基因轉殖小鼠乳皆無檢測出抑制血管收縮素轉化酶 (angiotensin-converting enzyme; ACE) 活性的能力。基於三品系基因轉殖小鼠的 AP1 mRNA 含量低落，我們懷疑 αLA 啟動子可能受到上下游沈默子 (Silencer) 抑制其轉錄活性。其中沈默子最有可能存在於 AP1 CDS 序列。此外，我們想了解為何 CRISPR/Cas9 的基因敲入率低落。透過比較四種基因轉殖動物的轉殖基因嵌插區周邊染色質結構，發現周邊序列具高相似性長片段不完全吻合的十字形 DNA 結構。推測大型十字形 DNA 是容易發生重組並被嵌入的序列。進一步製備 ROSA 的 5’ 端同源重組臂的反向重複序列，用以製造長片段十字形 DNA 結構。實驗結果顯示，反向重複組 (5’IR; 31.5%) 的基因敲入率高於非反向重複組 (KIR; 21.3%) 。由上述結果推測，同源重組中十字形 DNA 結構將會更易被辨識為模板，有助於外源 DNA 嵌入染色體組的效率。

Developing methods for mass-producing pharmaceutical protein is one of the important projects in the macromolecular drugs market. The mammary gland of mammals is a high-yield bioreactor with post-translational modification similar to the human body. The Anti-hypertension Peptide (AP1) had previously been isolated from Kefir in our lab. We produced mammary gland specific expressed AP1 transgenic mice using different transgenic techniques. The efficiency of genetically transgenic mice produced by the linear DNA group (8.7%) is similar to that of the CRISPR/Cas9 group (5.6%). Moreover, the survival rate of embryo transfer in the linear DNA group (39%) is higher than that of the CRISPR/Cas9 group (16%). And, we successfully produced three transgenic mouse strains FVB-Tg(αLA-AP1x1), FVB-Tg(αLA-Ap1x6)4, and ICR-Gt(ROSA)26Sorem(αLA-AP1x6)BM2, respectively. Unfortunately, the AP1 mRNA levels were low in the mammary glands of these transgenic mouse strains. Furthermore, the AP1 peptide was only detected by HPLC and LC/MS in the milk from FVB-Tg(αLA-Ap1x1). Then, angiotensin-converting enzyme (ACE) inhibition capabilities were undetectable in milk from these transgenic mouse strains. We thought that there might include silencers in AP1 CDS to inhibit the αLA promoter. On the other hand, we are interested in why CRISPR/Cas9 treatment is only low transgenic efficiency. We analyzed the chromatin structure around the transgenic insertion region of the four transgenic animals and found a cruciform DNA structure with high similarity in the surrounding sequence. We prepared the inverted repeat sequence of the 5-terminal homologous recombination arm of ROSA (5’IR) to create a cruciform DNA structure. The experimental results showed that the Knock-in rate of the 5’IR group (31.5%) was higher than that of the non-inverted repeat (KIR) group (21.3%). From the above results, we suggest that the structural characteristics of the template strand DNA play an important role in homologous recombination.