WO2022142092A1 - Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途 - Google Patents

Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途 Download PDF

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WO2022142092A1
WO2022142092A1 PCT/CN2021/095776 CN2021095776W WO2022142092A1 WO 2022142092 A1 WO2022142092 A1 WO 2022142092A1 CN 2021095776 W CN2021095776 W CN 2021095776W WO 2022142092 A1 WO2022142092 A1 WO 2022142092A1
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mice
fragment
gene
abcg4
abcg4 gene
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吴金美
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江苏科技大学
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  • the invention belongs to the field of animal genetic engineering and genetic modification, and in particular relates to the use of Abcg4 gene in constructing an animal model with a bidirectional change in obesity degree.
  • Sugars and lipids are essential nutrients for the human body, they both provide energy for cells and are also the basic structural components of cells. Glycolipid metabolism can be precisely regulated in healthy individuals, and abnormal glucose and lipid metabolism can cause many diseases including obesity, fatty liver, diabetes, atherosclerosis, cardiovascular and cerebrovascular diseases, and even cancer. Therefore, the homeostasis of glucose and lipid metabolism plays an important role in the health of the body.
  • Obesity is a complex disease caused by excessive accumulation of body fat, and obesity gene means that the protein encoded by the gene is a component of an appetite and energy balance regulation pathway, and the imbalance of this pathway directly or indirectly leads to the body. Fat accumulation and weight gain. Obesity not only affects the appearance of the human body, but also increases the risk of other diseases and health problems. In North America, two-thirds of American adults are classified as overweight or obese.
  • Obesity is affected by genetic factors, that is, genes.
  • genes genes.
  • GWASs genome-wide association studies
  • the number of genes found is generally large, and single gene analysis
  • its effect on obesity is not large, the results of different laboratories are also inconsistent, and there are few examples of mutual verification.
  • Fall T et al. used GWAS and meta-analyses to find that more than 75 loci were associated with obesity.
  • Another study reported that more than 500 genetic loci associated with obesity traits were found by genome-wide association analysis, but it is still difficult to pinpoint the causative gene within each locus.
  • mice have the most mature genetic manipulation and phenotypic analysis techniques, and are dominant in the breeding of animal models for various diseases.
  • the obesity animal models used for a long time have no difference in the degree of obesity between different genders, and there is no relevant report on the animal model with bidirectional changes in the degree of obesity between different genders.
  • the purpose of the present invention is to provide the use of the Abcg4 gene in constructing an animal model with a bidirectional change in the degree of obesity.
  • the first aspect of the present invention provides the use of the Abcg4 gene in constructing an animal model with a bidirectional change in the degree of obesity.
  • the use specifically refers to constructing an animal model by knocking out the Abcg4 gene in animals or inhibiting the expression of the Abcg4 gene.
  • the degree of obesity of the animal model is dependent on sex, and it is shown that with the same-sex wild-type littermate as a reference, females become obese and blood triglyceride levels decrease, and males become thinner and blood triglyceride levels increase. .
  • the animal is a mammal.
  • the mammal is a mouse.
  • the method for knocking out the mouse Abcg4 gene includes the following steps:
  • fragment I take the wild-type mouse genome as a template, amplify the fragment comprising the Abcg4 gene exon 6 to part 7 exon with primer pair 1, denoted as fragment I;
  • the nucleotide sequence of the primer pair 1 is:
  • fragment II Amplify the fragment containing exons 9-14 of Abcg4 gene with primer pair II, denoted as fragment II;
  • the nucleotide sequence of the primer pair II is:
  • the above-mentioned heterozygous progeny mice are backcrossed with C57BL/6J mice for N generation to generate a mouse germline deficient in important functional sites of the Abcg4 gene, and the heterozygotes are mated with each other to generate Abcg4 gene-deficient mice.
  • the present invention has the following beneficial effects:
  • the present invention uses gene knockout mice as a specific animal model for bidirectional changes between sexes of obesity or obesity-related diseases, mainly by reducing or completely knocking out at least one tissue or at least one cell type of mice.
  • the ATP-binding cassette transporter superfamily G subfamily gene No. 4 (abbreviated as Abcg4 gene) was prepared according to the method.
  • the obesity animal models used in the prior art have no difference in the degree of obesity between different genders.
  • the reverse change of obesity between genders found in the present invention is more suitable for individualized research and development of obesity and its related drugs.
  • this animal model can be used to screen out more effective and targeted drugs for the treatment of obesity, and it can also be used to screen out drugs that can target lower plasma triglyceride levels. ester drugs.
  • FIG. 1 is a schematic diagram of the strategy for constructing a mouse with deletion of important functional domains of the Abcg4 gene in Example 1.
  • FIG. 2 shows the results of confirming homologous recombination in mice by Southern hybridization in Example 2.
  • Figure 3 is a comparison of triglyceride content between Abcg4 gene-deficient mice and wild-type mice.
  • Figure 4 is a comparison of the paragonadal fat pad weight between Abcg4 gene-deficient mice and wild-type mice ( Figures A and B are different drawing methods of the same experiment, and the line in Figure B represents littermates).
  • Figure 5 is a comparison of lipid content between Abcg4 gene-deficient mice and wild-type mice ( Figure A and Figure B are different drawing methods of the same experiment, and the connecting line in Figure B represents littermates).
  • Figure 6 is a comparison of pancreas weights between Abcg4 gene-deficient mice and wild-type mice ( Figures A and B are different drawing methods of the same experiment, and the connecting line in Figure B represents littermates).
  • Figure 7 is the real-time quantitative PCR gene expression measurement in the brain of Abcg4 knockout female mice.
  • Figure 8 is the real-time quantitative PCR gene expression measurement in the brain of Abcg4 knockout male mice.
  • the method for knocking out the mouse Abcg4 gene of the present embodiment includes the following steps:
  • fragment I Using the wild-type mouse genome as a template, amplify the fragment comprising the Abcg4 gene exon 6 to part of the exon 7 with primer pair 1, which is denoted as fragment I;
  • the nucleotide sequence of the primer pair 1 is:
  • fragment II Amplify the fragment containing exons 9-14 of Abcg4 gene with primer pair II, denoted as fragment II;
  • the nucleotide sequence of the primer pair II is:
  • RII 5'-ATCAAAGAAGGACTGCCCCG-3' (as shown in SEQ ID NO.4);
  • fragment II Amplify the fragment containing exons 9-14 of Abcg4 gene with primer pair II, denoted as fragment II;
  • PCR amplification utilizes the "Expanded Long Template PCR System” kit of Roche (Expanded Long Template PCR System kit, Roche Applied Science, Indianapolis, IA, USA).
  • the program is: initial denaturation at 94°C for 3 minutes; 10 cycles of 94°C for 15 seconds, 65°C for 30 seconds, and 68°C for 8 minutes, followed by 94°C for 15 seconds, 65°C for 30 seconds, and 68°C for 8 minutes and 20 seconds 20 cycles were performed; final extension at 68°C for 7 minutes.
  • Genome identification method of positive ES cells The targeted recombinant vector was electroporated into C57B6/J mouse embryonic stem cells, and the transfected cells were selected with G418 for positive (positive) selection and ganciclovir for negative (negative) selection. The clones were screened by PCR and Southern blotting;
  • the genomic DNA of Abcg4 gene-deficient mice and the genomic DNA of wild-type mice obtained in Example 1 were digested with BgII enzyme, and probes were prepared for Southern hybridization detection.
  • the preparation method of the probe is as follows: firstly, the genomic DNA is amplified by PCR with primers int2F-2 and int5R.
  • the int2F-2 and int5R sequence information is as follows:
  • int2F 5'-CAGTACACAGGTTAAGCTAGGCAG-3' (as shown in SEQ ID NO.5);
  • int5R 5'-TCCATACACAGGCCTCGCCTAAGC-3' (as shown in SEQ ID NO. 6).
  • the PCR reaction system was: DNA template 10ng, primers 2ug each, Taq enzyme 1unit, 1 ⁇ PCR buffer, and the total reaction volume was 20ul.
  • PCR reaction conditions 94°C, 5 minutes; cycle: 94°C, 30 seconds, 60°C, 45 seconds, 72°C, 2 minutes, repeated 30 cycles; final: 72°C, 10 minutes; then stored at 4°C.
  • the PCR product was electrophoresed on agarose gel and purified by gel cutting, and then labeled with Roche's digoxigenin labeling kit (DIG-High Prime DNA Labeling and Detection System). After purification, it was used as a probe for Southern hybridization.
  • Roche's digoxigenin labeling kit DIG-High Prime DNA Labeling and Detection System
  • the target fragment (6.4 kb) with targeted insertion of the foreign fragment was larger than the wild-type (WT) fragment (4.5 kb).
  • Both parents of the knockout mice are heterozygous, and in the progeny produced by their mating, the result of Southern hybridization of Abcg4 knockout homozygotes (ko) has only a 6.4kb band (the third lane from the right). ), wild-type (wt) mice contain only one band of about 4.5kb (far right), and the other two mice are heterozygous (6.4kb and 4.5kb bands co-exist).
  • mice were anesthetized, their body weights were weighed and recorded, and the mice were dissected to take out the paragonadal fat pads, weighed and recorded.
  • the results are shown in Figure 4.
  • the ratio of the weight of the fat pad to the body weight of the mouse is the fat content (in percentage), and the results are shown in FIG. 5 .

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Abstract

提供了Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途，通过敲除动物体内Abcg4基因，或抑制Abcg4基因的表达，来构建动物模型。动物模型的肥胖程度依赖于性别，表现为以同窝生同性别野生型为参照，雌性变胖且血液甘油三酯含量降低，雄性变瘦且血液甘油三酯含量升高。该模型可用于研究肥胖症及相关疾病，并可用于筛选出更加有效、更有针对性的靶向治疗肥胖或治疗血浆甘油三酯升高的药物。

Description

Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途 技术领域
本发明属于动物基因工程和遗传修饰领域，具体的说，涉及Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途。
背景技术
糖和脂质是人体必需的营养物质，它们既为细胞提供能量同时也是细胞的基本结构成分。糖脂代谢在健康的个体中能被精准地调节，糖脂代谢异常则会引起包括肥胖症、脂肪肝、糖尿病、动脉粥样硬化、心脑血管疾病，甚至癌症等许多疾病。因此，糖脂代谢稳态平衡对机体的健康起着重要的作用。
肥胖是由身体的脂肪过量累积所产生的一种复杂的疾病，而肥胖基因，是指基因所编码蛋白质是一种食欲与能量平衡调节途径的组成部分，这种途径的失衡直接或者间接导致体脂肪的积累和体重增加。肥胖不仅仅影响人体的美观，更可以增加其它疾病和健康问题的患病风险。在北美洲，三分之二的美国成年人被归为超重或肥胖。
肥胖受遗传因素即基因的影响，近年来，大规模的基因组关联研究(GWA Ss,genome-wide association studies)方法应用于肥胖相关基因的鉴定，但一般找出的基因数量比较多，单个基因分析时其对肥胖的影响并不大，不同实验室的结果也不尽一致，能相互验证的例子较少。比如Fall T等运用GWAS和荟萃分析(meta-analyses)发现有超过75个基因位点与肥胖相关。最近，另有研究报道，通过全基因组关联分析发现了500多个与肥胖特征相关的基因位点，但每一个位点内精确定位致病基因仍很困难。
疾病动物模型可以为了解疾病发生发展机制，发展疾病预防和干预方法提供巨大帮助。作为哺乳动物，小鼠遗传操作和表型分析技术最为成熟，在各种疾病动物模型培育研究中占主导地位。但长期以来所使用的肥胖动物模型在不同的性别间其肥胖程度没有差异，且至今尚无不同性别间肥胖程度呈双向变化的动物模型的相关报道。
发明内容
为了克服现有技术中存在的问题，本发明的目的在于提供Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途。
为了实现上述目的，本发明采用如下技术方案：
本发明的第一个方面，提供Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途。
进一步的，所述用途具体是指通过敲除动物体内Abcg4基因或抑制Abcg4基因的表达，来构建动物模型。
更进一步的，所述动物模型的肥胖程度依赖于性别，表现为以同窝生同性别野生型为参照，雌性变胖且血液甘油三酯含量降低，雄性变瘦且血液甘油三酯含量升高。
更进一步的，所述动物为哺乳动物。
更进一步的，所述哺乳动物为小鼠。
更进一步的，敲除小鼠Abcg4基因的方法包括如下步骤：
S1、以野生型小鼠基因组为模板，以引物对I扩增包含Abcg4基因第6外显子至部分第7外显子的片段，记作片段I；
所述引物对I的核苷酸序列为：
FI:5’-TGGTGGATGGTGCAGATGAC-3’；
RI:5’-AAATCGGGGTGGTGCTTAGG3’；
以引物对II扩增包含Abcg4基因第9-14外显子的片段，记作片段II；
所述引物对II的核苷酸序列为：
FII:5’-AACTGACGAAGAAGCCGGAG3’；
RII：5’-ATCAAAGAAGGACTGCCCCG3’；
S2、将片段I插入到pCRII载体的eGFP编码区前，获得融合片段I，将融合片段I插入到靶向载体OSDUPDEL的多克隆位点A，将片段II插入到同一个靶向载体OSDUPDEL的多克隆位点B，获得靶向重组载体；
S3，上述重组载体经注射胚胎干细胞进行同源重组后，获得ES细胞打靶产物，对ES细胞打靶产物进行阳性克隆鉴定，筛选出正向敲除的ES细胞；
S4，将S3筛选出的ES细胞注射到C57BL/6J小鼠囊胚中并植入假孕母鼠体内，获得具有种系传递能力的杂合子代小鼠；
S5，将上述杂合子代小鼠与C57BL/6J小鼠进行N代的回交，产生Abcg4基因重要功能部位缺陷鼠种系，杂合子相互交配，生成Abcg4基因缺陷型小鼠。
更进一步的，S5中，N≥16。
与现有技术相比，本发明具有如下有益效果：
1、本发明将基因敲除的小鼠用作肥胖或肥胖相关疾病的性别间双向变化的特异动物模型，主要通过减少或完全敲除小鼠的至少一种组织或至少一种细胞类型中的ATP结合盒转运蛋白超家族G亚族中第4号成员基因(简称Abcg4基因)的量的方法来制作。
2、现有技术所使用的肥胖动物模型在不同的性别间其肥胖程度没有差异，本发明所发现的性别间肥胖的反向变化，更适合于肥胖及其相关药物的个性化研究与开发。
3、在肥胖及其糖尿病等相关疾病的研究上，可以使用该动物模型筛选出更加有效、更有针对性的靶向治疗肥胖的药物，也可以利用其筛选出可以靶向降低血浆中甘油三酯的药物。
附图说明
图1为实施例1构建Abcg4基因重要功能域缺失鼠的策略示意图。
图2为实施例2用Southern杂交证实小鼠同源重组的结果。
图3为Abcg4基因缺陷型小鼠和野生型小鼠甘油三酯含量比较。
图4为Abcg4基因缺陷型小鼠和野生型小鼠性腺旁脂肪垫重量比较(图A和图B为同一个实验的不同画法，图B中连线表示同窝生)。
图5为Abcg4基因缺陷型小鼠和野生型小鼠含脂率比较(图A和图B为同一个实验的不同画法，图B中连线表示同窝生)。
图6为Abcg4基因缺陷型小鼠和野生型小鼠胰腺重量比较(图A和图B为同一个实验的不同画法，图B中连线表示同窝生)。
图7为Abcg4基因敲除雌鼠鼠脑实时定量PCR基因表达测定。
图8为Abcg4基因敲除雄鼠脑实时定量PCR基因表达测定。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商 业途径得到。
实施例1
参见图1，本实施例的敲除小鼠Abcg4基因的方法，包括如下步骤：
以野生型小鼠基因组为模板，以引物对I扩增包含Abcg4基因第6外显子至部分第7外显子的片段，记作片段I；
所述引物对I的核苷酸序列为：
FI:5’-TGGTGGATGGTGCAGATGAC-3’(如SEQ ID NO.1所示)；
RI:5’-AAATCGGGGTGGTGCTTAGG-3’(如SEQ ID NO.2所示)；
以引物对II扩增包含Abcg4基因第9-14外显子的片段，记作片段II；
所述引物对II的核苷酸序列为：
FII:5’-AACTGACGAAGAAGCCGGAG-3’(如SEQ ID NO.3所示)；
RII：5’-ATCAAAGAAGGACTGCCCCG-3’(如SEQ ID NO.4所示)；
以引物对II扩增包含Abcg4基因第9-14外显子的片段，记作片段II；
S2、将片段I插入到pCRII载体的eGFP编码区前，获得融合片段I，将融合片段I插入到靶向载体OSDUPDEL(OSDupDel Gene Targeting Vector-neo)载体的多克隆位点A(MCSA)，将片段II插入到同一个靶向载体OSDUPDEL载体的多克隆位点B(MCSB)，获得靶向重组载体；
上述PCR扩增利用Roche的“扩展长模板PCR系统”试剂盒(Expanded Long Template PCR System kit，Roche Applied Science,Indianapolis,IA,USA)。
程序为：94℃初始变性3分钟；先以94℃ 15秒，65℃ 30秒，68℃ 8分钟，进行10个循环，然后以94℃ 15秒，65℃ 30秒，68℃ 8分20秒进行20个循环；最终68℃延伸7分钟。
S3，上述靶向重组载体经注射胚胎干细胞进行同源重组后，获得ES细胞打靶产物，对ES细胞打靶产物进行阳性克隆鉴定，筛选出正向敲除的ES细胞；
阳性ES细胞基因组鉴定方法：将靶向重组载体电转到C57B6/J小鼠胚胎干细胞中，转染细胞用G418作正向(阳性)选择，用更昔洛韦作负向(阴性)选择，存活的克隆通过PCR和Southern印迹法进行筛选；
S4，将S3筛选出的ES细胞注射到C57BL/6J小鼠囊胚中并植入假孕母鼠体内，获得具有种系传递能力的杂合子代小鼠；
S5，将上述杂合子代小鼠与C57BL/6J小鼠进行16代的回交，产生Abcg4基因重要功能部位缺陷鼠种系，杂合子相互交配，生成Abcg4基因缺陷型小鼠。
实施例2
Abcg4基因缺陷型小鼠基因型鉴定
用Southern杂交证实同源重组的正确性，包括如下步骤：
先用BgII酶消化实施例1获得的Abcg4基因缺陷型小鼠的基因组DNA以及野生型小鼠基因组DNA，制作探针进行Southern杂交检测。
所述探针的制备方法如下：先用引物int2F-2和int5R对基因组DNA进行PCR扩增。int2F-2和int5R序列信息如下：
int2F：5’-CAGTACACAGGTTAAGCTAGGCAG-3’(如SEQ ID NO.5所示)；
int5R：5’-TCCATACACAGGCCTCGCCTAAGC-3’(如SEQ ID NO.6所示)。
PCR反应体系为：DNA模版10ng，引物各2ug，Taq酶1unit，1×PCR buffer，反应总体积为20ul。
PCR反应条件：94℃，5分钟；循环：94℃，30秒，60℃，45秒，72℃，2分钟，重复30个循环；最后：72℃，10分钟；然后于4℃保存。PCR产物琼脂糖凝胶电泳并切胶纯化后，用罗氏的地高辛标记试剂盒(DIG-High Prime DNA Labeling andDetection System)进行标记，纯化后作为探针用于Southern杂交。
结果如图2所示，具有靶向插入外源片段的靶片段(6.4kb)大于野生型(WT)片段(4.5kb)。基因敲除鼠父母双方均为杂合子，使其交配所产生的子代中，Abcg4敲除的纯合子(ko)Southern杂交的结果仅有6.4kb大小的一条带(右边数起第3个泳道)，野生型(wt)小鼠仅含一条约4.5kb的一条带(最右边)，其它两个小鼠为杂合型(6.4kb和4.5kb的带子同时存在)。此实验证明，通过基因组的靶向同源重组所产生的Abcg4基因重要功能域敲除的小鼠后代，其基因型及Southern杂交的片段大小与理论值相符，证明了靶向敲除的正确性。(注:最左边的分子量标记位置用溴化乙锭进行凝胶染色来估算。)
实施例3
利用肥胖症指标表征Abcg4基因缺陷型小鼠
以下结果均是以出生后4周同窝生Abcg4基因缺陷型小鼠和野生型小鼠进行的比较。
(1)甘油三酯含量分析
分析过程及结果：使用Boehringer Mannheim公司的甘油三酯分析试剂盒，根据其说明书操作，结果如图3所示。
(2)性腺旁脂肪垫分析
分析过程及结果：小鼠麻醉，称取并记录体重，解剖小鼠取出性腺旁脂肪垫，称重并记录，结果如图4所示。脂肪垫重量与该小鼠的体重之比即为含脂率(以百分比计)，结果如图5所示。
(3)胰腺重量分析
分析过程及结果:解剖小鼠取出胰腺，并记录，胰腺重量结果如图6所示。
实施例4
利用小鼠脑内糖脂代谢相关基因的表达，从分子水平侧面验证了Abcg4基因缺陷型小鼠在其脂肪垫/体重百分比变化的性别“二态性”
(1)Abcg4基因敲除鼠的雌性鼠与同窝生的野生型雌鼠相比，其脑内与糖脂代谢相关的系列基因的表达情况，以实验当时连接实时定量PCR仪的电脑软件直接输出的结果(print screen)复制而来，代表没有对图片进行任何人为的加工与修饰。野生型小鼠基因的相对表达量设为1，如图7所示。
(2)基因敲除雄鼠与同窝生野生型小鼠相比，脑内的糖脂代谢相关基因表达情况如图8所示。
从以上不同性别Abcg4基因敲除小鼠脑内基因表达结果可以看出，雌性与雄性基因敲除鼠高表达与低表达的基因几乎是相反的。这与本实验观察到的雌性小鼠性腺旁脂肪垫重量与体重之比率比野生型小鼠增加(变胖)，而雄鼠该比例降低(变瘦)的表型相吻合。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若针对本发明的这些修改和变型属于本发明权利要 求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。

Claims (7)

  1. Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途。
  2. 根据权利要求1所述的用途，其特征在于，所述用途具体是指通过敲除动物体内Abcg4基因或抑制Abcg4基因的表达，来构建动物模型。
  3. 根据权利要求2所述的用途，其特征在于，所述动物模型的肥胖程度依赖于性别，表现为以同窝生同性别野生型为参照，雌性变胖且血液甘油三酯含量降低，雄性变瘦且血液甘油三酯含量升高。
  4. 根据权利要求3所述的用途，其特征在于，所述动物为哺乳动物。
  5. 根据权利要求4所述的用途，其特征在于，所述哺乳动物为小鼠。
  6. 根据权利要求5所述的用途，其特征在于，敲除小鼠Abcg4基因的方法包括如下步骤：
    S1、以野生型小鼠基因组为模板，以引物对I扩增包含Abcg4基因第6外显子至部分第7外显子的片段，记作片段I；
    所述引物对I的核苷酸序列为：
    FI:5’-TGGTGGATGGTGCAGATGAC-3’；
    RI:5’-AAATCGGGGTGGTGCTTAGG3’；
    以引物对II扩增包含Abcg4基因第9-14外显子的片段，记作片段II；
    所述引物对II的核苷酸序列为：
    FII:5’-AACTGACGAAGAAGCCGGAG3’；
    RII：5’-ATCAAAGAAGGACTGCCCCG3’；
    S2、将片段I插入到pCRII载体的eGFP编码区前，获得融合片段I，将融合片段I插入到靶向载体OSDUPDEL的多克隆位点A，将片段II插入到同一个靶向载体OSDUPDEL的多克隆位点B，获得靶向重组载体；
    S3，上述重组载体经注射胚胎干细胞进行同源重组后，获得ES细胞打靶产物，对ES细胞打靶产物进行阳性克隆鉴定，筛选出正向敲除的ES细胞；
    S4，将S3筛选出的ES细胞注射到C57BL/6J小鼠囊胚中并植入假孕母鼠体内，获得具有种系传递能力的杂合子代小鼠；
    S5，将上述杂合子代小鼠与C57BL/6J小鼠进行N代的回交，产生Abcg4基因重要功能部位缺陷鼠种系，杂合子相互交配，生成Abcg4基因缺陷型小鼠。
  7. 根据权利要求6所述的用途，其特征在于，S5中，N≥16。
PCT/CN2021/095776 2020-12-28 2021-05-25 Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途 WO2022142092A1 (zh)

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