ob/ob (Lep^ob)

提前一周单笼饲养实验前一周将小鼠改为单笼饲养。称量体重逐只称量小鼠体重，用于按体重计算给糖体积。禁食6小时禁食六小时（06:00:00）。测定基线血糖禁食结束后，先用血糖仪测定血糖作为基线。腹腔注射葡萄糖按2 g/kg体重腹腔注射葡萄糖：将2 g D-葡萄糖溶于10 ml生理盐水配成20%葡萄糖贮备液，按每克体重10 ul给予。系列采血测定血糖于注射葡萄糖后15、30、45、60、90和120分钟采集血样，用血糖仪测定血糖。

禁食 Fasting begins first thing in the morning (around 9am). Fast mice for 4-6 hours before OGTT begins. Transfer mice to clean cage and wire top. Keep water bottles during the fasting period. 1. If using High Fat diet (HFD), save food to give back at the end of the OGTT. 1. mark tails with a sharpie for easier identification during OGTT. 口服葡萄糖负荷 Weigh mouse to obtain body weight for dose calculations. Calculate bolus dose of glucose (**1g/kg**). Weight of mouse (g) x 2 = ul sterile glucose (50% dextrose) solution *If using HFD for 4 weeks or more, use 0.5g/kg (weight of mouse (g) x 1 = ul sterile glucose (50% dextrose) solution) 口服葡萄糖负荷 Prepare syringes using 1cc syringe and 18G oral gavage needle. Load all syringes with calculated glucose dose and position in front of home cage. 口服葡萄糖负荷 Prior to the delivery of the sterile glucose (50% dextrose) solution, a time “zero” blood glucose level must be measured. Restrain mouse and clean tail with 70% ethanol/gauze. Extract a small amount of blood from the tail vein onto One Touch Ultra blood glucose strips used with the One Touch Ultra 2 blood glucose monitoring system. Blood can be collected into microvette tubes that can be used to extract plasma which can be assayed for plasma insulin (see step 9 for plasma collection). Microvette tubes are blood collection tubes with a capillary. Tap tube until all the blood is collected in the tube. Place on ice until plasma collection. 口服葡萄糖负荷 Administer the correct dose of sterile glucose (50% dextrose) solution using the syringe and gavage needle. - Restraint of the Mouse, avoiding an excessively tight grip of the mouse, as this will induce a panic response. Ensure that the mouse cannot bite you and cannot move its head. - Gravity alone should be used to move the cannula down the esophagus. While gliding the cannula, rotate slightly as the cannula goes past the epiglottis and into the esophagus.The mouse should swallow the cannula, and this can be observed. - Inject the fluid slowly. - The cannula is then gently removed and the animal is returned to its cage. 口服葡萄糖负荷 Begin timing from the point of successfully delivering the sterile glucose (50% dextrose) solution. 口服葡萄糖负荷 Test and record the blood glucose level (see step 4) at 15, 30, 60, 90 and 120 minutes after the initial glucose delivery. Record each value and the condition of the animal on the Glucose Tolerance Test record sheet. Extract a small amount of blood from the tail vein at 15, 30, 60 and 120 minutes for plasma; skip blood collection at 90 minutes to ensure not to exceed 0.007 ml blood/ g body weight. 口服葡萄糖负荷 Return food and environmental enrichments to cages. 血浆采集 Place tubes into pre-chilled centrifuge. Centrifguge for 2 mins at 2000 rpm. 00:02:00 4 °C 血浆采集 Remove the callipary-lid and dispose in biohazard bin. Place the base lid and seal the microvette (click position). 血浆采集 Centrifuge at 4°C for 10 mins at 10 000 rpm. 00:10:00 4 °C 血浆采集 Using a pipette, transfer the plasma (top transparent layer) to a clean and labelled tube. Store plasma samples at -20°C until assayed. 血浆胰岛素测定 Assay plasma insulin with the STELLUX Chemi Rodent Insulin ELISA kit (ALPCO #80-INSMR-CH01) according to the manufacturer's protocol. Include the kit standards and quality controls in each assay and read the plate by the kit's chemiluminescence procedure.

禁食 Fasting begins first thing in the morning (around 9am). Fast mice for 4-6 hours before ITT begins. Transfer mice to clean cage and wire top. Keep water bottles during the fasting period. 1. If using High Fat diet (HFD), save food to give back at the end of the ITT. 1. Mark tails with a sharpie for easier identification during ITT. 胰岛素溶液配制 Stock 1: 50ul of 100U/ml Humulin R into 4950ul of cold sterile 1X PBS. Invert solution 3-4 times to mix. Stock 2: 1ml of Stock 1 into 9ml of cold sterile 1X PBS. Invert solution 3-4 times to mix. Keep on ice until use 胰岛素负荷 Weigh mouse to obtain body weight for dose calculations. Calculate bolus dose of insulin (**1-1.2 units/kg**). Weight of mouse (g) x 10 = ul insulin stock 2 solution 胰岛素负荷 Prepare syringes using 1cc syringe and 26G needle. Load all syringes with calculated insulin dose and position in front of home cage. 胰岛素负荷 Prior to the delivery of the insulin solution, a time “zero” blood glucose level must be measured. Restrain mouse and clean tail with 70% ethanol/gauze. Extract a small amount of blood from the tail vein onto One Touch Ultra blood glucose strips used with the One Touch Ultra 2 blood glucose monitoring system. 胰岛素负荷 Administer the correct dose of Insulin solution using an IP (intraperitoneal) injection method. - Restrain the mouse. Then place the mouse is in dorsal recumbency with the head down allowing the viscera to move cranially. - Using the bend of the knee and the ventral abdominal midline as landmarks, the needle is inserted half way between the midline and lateral side of the animal at the natural bend of the knee. Insert the needle at a 30 to 45 degree angle into either the left or right lower abdominal quadrant. - Prior to injection, aspirate to make sure the needle has not penetrated a blood vessel, the intestines or the urinary bladder. When aspirating the syringe, you should see an air bubble in the hub of the needle, and not any form of fluid. - Inject and return mouse to cage. 胰岛素负荷 Begin timing from the point of successfully delivering the insulin solution. 胰岛素负荷 Test and record the blood glucose level at 15, 30, 60, 90, and 120 minutes after the insulin injection. Record each value and the condition of the animal on the Insulin Tolerance Test record sheet. 胰岛素负荷 Return food and environmental enrichments to cages.

Calibrate Cobas for Glucose analysis by running a multi analyte standard and two control serum. Calibrate Cobas for Glucose analysis by running a multi analyte standard and two control serum. Sample Handling as performed by the Cobas Mira Plus. Sample Handling as performed by the Cobas Mira Plus. a) Pipette 3 µL of sample into cuvette. b) Absorbance is measured at 340 nm. c) Add 100 µL of Glucose liquid reagent. d) Mixture is incubated at 37ºC for 10 minutes. e) Absorbance is measured at 340 nm. Change in absorbance is calculated.

脱蜡（Dewaxing）石蜡切片置于二甲苯中脱蜡 3 次，每次 00:03:00。复水（Rehydration）石蜡切片依次置于 100% 乙醇中 3 次、每次 00:03:00，再置于 95% 乙醇中 1 次、00:03:00，随后用水漂洗。苏木素核染色（Hematoxylin staining）石蜡切片以苏木素染液染色 00:00:20，随后在流水下冲洗 00:01:00。分化与返蓝（Differentiation and bluing）石蜡切片置于返蓝液中 00:01:00，随后在流水下冲洗 00:01:00；此步使细胞核呈现蓝色。伊红染色（Eosin staining）石蜡切片以伊红复染 00:00:10；此步使细胞质成分与结缔组织纤维呈现粉红色。脱水与透明（Dehydration and Clearing）石蜡切片依次置于 95% 乙醇中 00:02:00、100% 乙醇中 2 次、每次 00:02:00，随后置于二甲苯中 2 次、每次 00:02:00。封片（Mounting）石蜡切片以 Permount 封片介质加盖玻片封固。

Model identity

Model name: ob/ob (Lep^ob)
Species: mouse
Taxonomy ID: 10090
Strain: B6.Cg-Lep<ob>/J
Genetic background: 在 C57BL/6 背景上携带 6 号染色体 Lep 基因 Lep<ob> 自发突变的同源导入（congenic）品系，品系名前缀 B6.Cg 即此遗传构成；参考记录的同用名包括 B6.V-Lep<ob>/J 与 C57BL/6J-Lep<ob>，对应参考资源编号 JAX 000632（常用名 B6 ob）。以上为公开参考品系记录的遗传背景，不代表公司实际动物来源与群落背景。
Genotype / zygosity: Lep<ob> 纯合突变（ob/ob）：瘦素（Lep）等位基因经 C→T 点突变产生精氨酸密码子 105 无义突变（p.R105*），16 kDa 瘦素蛋白缺如；遗传方式为隐性。
Modification locus: 6 号染色体 Lep 基因座（MGI:104663，约 12.3 cM）：等位基因 Lep<ob>（MGI:1856424，自发性点突变，Null/knockout）。
Allele: Lep<ob>（等位基因名 leptin; obese；MGI:1856424；自发性突变，Null/knockout）。
MGI ID: MGI:2161625

Biological characteristics

Phenotype stage: 3–4 周龄起肥胖可识别，随后出现糖耐量异常和胰岛素抵抗；具体时点与饮食、性别和来源品系有关。
Research questions: 瘦素蛋白缺失在摄食、体脂与血糖稳态调控中的作用；Lep<ob> 隐性无义突变纯合如何形成肥胖与高血糖表型；ob 位点（leptin）及其人同源基因的克隆与功能研究。
Limitations: 单基因瘦素缺陷不能代表多基因人类肥胖；C57BL/6 背景、饮食和肠道菌群影响表型严重度；不同来源 ob/ob 品系的代谢表型存在差异，不能把单一研究的时间点当作统一病程。

Experimental methods

Experimental protocols

小鼠腹腔注射葡萄糖耐量检测（IPGTT）
Intraperitoneal glucose tolerance test (IPGTT) in mice: single-house for one week, weigh, fast for 6 hours, measure baseline glucose, inject 2 g/kg body weight of 20% D-glucose intraperitoneally, then collect blood at 15 to 120 minutes after injection. Used to assess glucose clearance in obesity and glucose-metabolism models.
小鼠口服葡萄糖耐量试验（OGTT）
This protocol adopts the protocols.io Oral Glucose Tolerance Test in Mouse v1. Mice are fasted for 4–6 hours, weighed, and given oral glucose at 1 g/kg (0.5 g/kg after at least 4 weeks of high-fat diet). Blood glucose is measured before and at 15, 30, 60, 90 and 120 minutes after the load, with tail-vein plasma collected at selected time points for insulin measurement. The record describes the assay procedure, not executed disease-model results.
小鼠胰岛素耐量试验（ITT）
This protocol adopts the protocols.io Insulin Tolerance Test in Mouse v1. Mice are fasted for 4–6 hours, weighed, and given insulin intraperitoneally at 1–1.2 U/kg. Blood glucose is measured before and at 15, 30, 60, 90 and 120 minutes after injection. The measurement is a glucose assay (OBI:2100020); insulin administration, dose and time points belong to this concrete protocol and are not presented as an exact OBI insulin-tolerance-test term.
血液葡萄糖浓度酶法测定
Enzymatic blood glucose quantification by coupled hexokinase and glucose-6-phosphate dehydrogenase reactions, using absorbance change at 340 nm on the Cobas Mira Plus.
小鼠口服葡萄糖耐量试验（OGTT）
This protocol adopts the protocols.io Oral Glucose Tolerance Test in Mouse v1. Mice are fasted for 4–6 hours, weighed, and given oral glucose at 1 g/kg (0.5 g/kg after at least 4 weeks of high-fat diet). Blood glucose is measured before and at 15, 30, 60, 90 and 120 minutes after the load, with tail-vein plasma collected at selected time points for insulin measurement. The record describes the assay procedure, not executed disease-model results.
小鼠胰岛素耐量试验（ITT）
This protocol adopts the protocols.io Insulin Tolerance Test in Mouse v1. Mice are fasted for 4–6 hours, weighed, and given insulin intraperitoneally at 1–1.2 U/kg. Blood glucose is measured before and at 15, 30, 60, 90 and 120 minutes after injection. The measurement is a glucose assay (OBI:2100020); insulin administration, dose and time points belong to this concrete protocol and are not presented as an exact OBI insulin-tolerance-test term.
Hematoxylin and Eosin (H&E) staining for histopathology
Hematoxylin and eosin (H&E) staining of fixed, paraffin-embedded tissue sections (4 μm). The workflow covers dewaxing, rehydration, hematoxylin nuclear staining, bluing, eosin counterstaining, dehydration and clearing, and coverslipping, yielding blue-purple nuclei and pink cytoplasm and connective tissue fibers for light-microscopic assessment of cellular and tissue morphology.
小鼠腹腔注射葡萄糖耐量检测（IPGTT）
Intraperitoneal glucose tolerance test (IPGTT) in mice: single-house for one week, weigh, fast for 6 hours, measure baseline glucose, inject 2 g/kg body weight of 20% D-glucose intraperitoneally, then collect blood at 15 to 120 minutes after injection. Used to assess glucose clearance in obesity and glucose-metabolism models.
小鼠口服葡萄糖耐量试验（OGTT）
This protocol adopts the protocols.io Oral Glucose Tolerance Test in Mouse v1. Mice are fasted for 4–6 hours, weighed, and given oral glucose at 1 g/kg (0.5 g/kg after at least 4 weeks of high-fat diet). Blood glucose is measured before and at 15, 30, 60, 90 and 120 minutes after the load, with tail-vein plasma collected at selected time points for insulin measurement. The record describes the assay procedure, not executed disease-model results.
小鼠腹腔注射葡萄糖耐量检测（IPGTT）
Intraperitoneal glucose tolerance test (IPGTT) in mice: single-house for one week, weigh, fast for 6 hours, measure baseline glucose, inject 2 g/kg body weight of 20% D-glucose intraperitoneally, then collect blood at 15 to 120 minutes after injection. Used to assess glucose clearance in obesity and glucose-metabolism models.
小鼠胰岛素耐量试验（ITT）
This protocol adopts the protocols.io Insulin Tolerance Test in Mouse v1. Mice are fasted for 4–6 hours, weighed, and given insulin intraperitoneally at 1–1.2 U/kg. Blood glucose is measured before and at 15, 30, 60, 90 and 120 minutes after injection. The measurement is a glucose assay (OBI:2100020); insulin administration, dose and time points belong to this concrete protocol and are not presented as an exact OBI insulin-tolerance-test term.
Hematoxylin and Eosin (H&E) staining for histopathology
Hematoxylin and eosin (H&E) staining of fixed, paraffin-embedded tissue sections (4 μm). The workflow covers dewaxing, rehydration, hematoxylin nuclear staining, bluing, eosin counterstaining, dehydration and clearing, and coverslipping, yielding blue-purple nuclei and pink cytoplasm and connective tissue fibers for light-microscopic assessment of cellular and tissue morphology.
小鼠口服葡萄糖耐量试验（OGTT）
This protocol adopts the protocols.io Oral Glucose Tolerance Test in Mouse v1. Mice are fasted for 4–6 hours, weighed, and given oral glucose at 1 g/kg (0.5 g/kg after at least 4 weeks of high-fat diet). Blood glucose is measured before and at 15, 30, 60, 90 and 120 minutes after the load, with tail-vein plasma collected at selected time points for insulin measurement. The record describes the assay procedure, not executed disease-model results.
小鼠口服葡萄糖耐量试验（OGTT）
This protocol adopts the protocols.io Oral Glucose Tolerance Test in Mouse v1. Mice are fasted for 4–6 hours, weighed, and given oral glucose at 1 g/kg (0.5 g/kg after at least 4 weeks of high-fat diet). Blood glucose is measured before and at 15, 30, 60, 90 and 120 minutes after the load, with tail-vein plasma collected at selected time points for insulin measurement. The record describes the assay procedure, not executed disease-model results.
小鼠口服葡萄糖耐量试验（OGTT）
This protocol adopts the protocols.io Oral Glucose Tolerance Test in Mouse v1. Mice are fasted for 4–6 hours, weighed, and given oral glucose at 1 g/kg (0.5 g/kg after at least 4 weeks of high-fat diet). Blood glucose is measured before and at 15, 30, 60, 90 and 120 minutes after the load, with tail-vein plasma collected at selected time points for insulin measurement. The record describes the assay procedure, not executed disease-model results.
小鼠口服葡萄糖耐量试验（OGTT）
This protocol adopts the protocols.io Oral Glucose Tolerance Test in Mouse v1. Mice are fasted for 4–6 hours, weighed, and given oral glucose at 1 g/kg (0.5 g/kg after at least 4 weeks of high-fat diet). Blood glucose is measured before and at 15, 30, 60, 90 and 120 minutes after the load, with tail-vein plasma collected at selected time points for insulin measurement. The record describes the assay procedure, not executed disease-model results.
小鼠腹腔注射葡萄糖耐量检测（IPGTT）
Intraperitoneal glucose tolerance test (IPGTT) in mice: single-house for one week, weigh, fast for 6 hours, measure baseline glucose, inject 2 g/kg body weight of 20% D-glucose intraperitoneally, then collect blood at 15 to 120 minutes after injection. Used to assess glucose clearance in obesity and glucose-metabolism models.
小鼠胰岛素耐量试验（ITT）
This protocol adopts the protocols.io Insulin Tolerance Test in Mouse v1. Mice are fasted for 4–6 hours, weighed, and given insulin intraperitoneally at 1–1.2 U/kg. Blood glucose is measured before and at 15, 30, 60, 90 and 120 minutes after injection. The measurement is a glucose assay (OBI:2100020); insulin administration, dose and time points belong to this concrete protocol and are not presented as an exact OBI insulin-tolerance-test term.
Hematoxylin and Eosin (H&E) staining for histopathology
Hematoxylin and eosin (H&E) staining of fixed, paraffin-embedded tissue sections (4 μm). The workflow covers dewaxing, rehydration, hematoxylin nuclear staining, bluing, eosin counterstaining, dehydration and clearing, and coverslipping, yielding blue-purple nuclei and pink cytoplasm and connective tissue fibers for light-microscopic assessment of cellular and tissue morphology.

Reagents and materials

D-葡萄糖（D-glucose）
葡萄糖负荷溶质
0.9% 氯化钠溶液（生理盐水）
葡萄糖负荷液的溶剂/载体
血糖试纸
血糖测定
D-葡萄糖（D-glucose）
口服葡萄糖负荷
70%乙醇
尾静脉采血部位消毒
STELLUX Chemi Rodent Insulin ELISA（ALPCO 80-INSMR-CH01）
血浆胰岛素定量
血糖试纸
血糖测定
胰岛素（insulin）
胰岛素负荷
70%乙醇
尾静脉采血部位消毒
PBS 磷酸盐缓冲液
胰岛素稀释液
葡萄糖酶法检测试剂（Cliniqa R84682）
葡萄糖酶法反应试剂
多分析物校准品（Cliniqa R60010）
分析仪校准
定值质控血清1（Cliniqa R83082）
质控血清1
定值质控血清2（Cliniqa R83083）
质控血清2
STELLUX Chemi Rodent Insulin ELISA（ALPCO 80-INSMR-CH01）
血浆胰岛素定量
70%乙醇
尾静脉采血部位消毒
D-葡萄糖（D-glucose）
口服葡萄糖负荷
血糖试纸
血糖测定
PBS 磷酸盐缓冲液
胰岛素稀释液
70%乙醇
尾静脉采血部位消毒
胰岛素（insulin）
胰岛素负荷
血糖试纸
血糖测定
Permount 组织学封片剂（Fisher SP15）
封片介质
Epredia Signature Series 伊红 Y 染液（7111）
细胞质与结缔组织纤维复染
Epredia Signature Series 返蓝液（7301）
返蓝（使细胞核转蓝）
Epredia Signature Series 苏木素染液（7221）
细胞核染色
自来水（组织学染色用）
复水后漂洗用水（后续各流水冲洗共用）
95% 乙醇溶液
复水梯度溶剂
Ethanol
复水梯度溶剂（去除二甲苯）
组织学二甲苯（混合异构体）
脱蜡剂
0.9% 氯化钠溶液（生理盐水）
葡萄糖负荷液的溶剂/载体
D-葡萄糖（D-glucose）
葡萄糖负荷溶质
血糖试纸
血糖测定
D-葡萄糖（D-glucose）
口服葡萄糖负荷
70%乙醇
尾静脉采血部位消毒
STELLUX Chemi Rodent Insulin ELISA（ALPCO 80-INSMR-CH01）
血浆胰岛素定量
D-葡萄糖（D-glucose）
葡萄糖负荷溶质
0.9% 氯化钠溶液（生理盐水）
葡萄糖负荷液的溶剂/载体
血糖试纸
血糖测定
胰岛素（insulin）
胰岛素负荷
70%乙醇
尾静脉采血部位消毒
PBS 磷酸盐缓冲液
胰岛素稀释液
组织学二甲苯（混合异构体）
脱蜡剂
Ethanol
复水梯度溶剂（去除二甲苯）
95% 乙醇溶液
复水梯度溶剂
自来水（组织学染色用）
复水后漂洗用水（后续各流水冲洗共用）
Epredia Signature Series 苏木素染液（7221）
细胞核染色
Epredia Signature Series 返蓝液（7301）
返蓝（使细胞核转蓝）
Epredia Signature Series 伊红 Y 染液（7111）
细胞质与结缔组织纤维复染
Permount 组织学封片剂（Fisher SP15）
封片介质
STELLUX Chemi Rodent Insulin ELISA（ALPCO 80-INSMR-CH01）
血浆胰岛素定量
70%乙醇
尾静脉采血部位消毒
D-葡萄糖（D-glucose）
口服葡萄糖负荷
血糖试纸
血糖测定
血糖试纸
血糖测定
D-葡萄糖（D-glucose）
口服葡萄糖负荷
70%乙醇
尾静脉采血部位消毒
STELLUX Chemi Rodent Insulin ELISA（ALPCO 80-INSMR-CH01）
血浆胰岛素定量
STELLUX Chemi Rodent Insulin ELISA（ALPCO 80-INSMR-CH01）
血浆胰岛素定量
70%乙醇
尾静脉采血部位消毒
D-葡萄糖（D-glucose）
口服葡萄糖负荷
血糖试纸
血糖测定
血糖试纸
血糖测定
D-葡萄糖（D-glucose）
口服葡萄糖负荷
70%乙醇
尾静脉采血部位消毒
STELLUX Chemi Rodent Insulin ELISA（ALPCO 80-INSMR-CH01）
血浆胰岛素定量
0.9% 氯化钠溶液（生理盐水）
葡萄糖负荷液的溶剂/载体
D-葡萄糖（D-glucose）
葡萄糖负荷溶质
PBS 磷酸盐缓冲液
胰岛素稀释液
70%乙醇
尾静脉采血部位消毒
胰岛素（insulin）
胰岛素负荷
血糖试纸
血糖测定
组织学二甲苯（混合异构体）
脱蜡剂
Ethanol
复水梯度溶剂（去除二甲苯）
95% 乙醇溶液
复水梯度溶剂
自来水（组织学染色用）
复水后漂洗用水（后续各流水冲洗共用）
Epredia Signature Series 苏木素染液（7221）
细胞核染色
Epredia Signature Series 返蓝液（7301）
返蓝（使细胞核转蓝）
Epredia Signature Series 伊红 Y 染液（7111）
细胞质与结缔组织纤维复染
Permount 组织学封片剂（Fisher SP15）
封片介质

References

Model consultation