,, 张平, 张晓枫, 李兴, 白宇, 贾克荣, 郭晓东, 张豪, 马晓燕, 仓明, 刘东军, 郭旭东内蒙古大学生命科学学院,实验动物研究中心,呼和浩特 010070Construction of FGF21 knockout mouse models by the CRISPR/Cas9 system
Xu Liu,, Ping Zhang, Xiaofeng Zhang, Xing Li, Yu Bai, Kerong Jia, Xiaodong Guo, Hao Zhang, Xiaoyan Ma, Ming Cang, Dongjun Liu, Xudong GuoThe Research Center for Laboratory Animal Sciense, College of Life Science, Inner Mongolia University, Hohhot 010070, China第一联系人:
收稿日期:2017-05-16修回日期:2017-12-22网络出版日期:--
| 基金资助: |
Received:2017-05-16Revised:2017-12-22Online:--
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刘旭, 张平, 张晓枫, 李兴, 白宇, 贾克荣, 郭晓东, 张豪, 马晓燕, 仓明, 刘东军, 郭旭东. 利用CRISPR/Cas9系统构建FGF21基因敲除小鼠模型. 遗传[J], 2018, 40(1): 66-74 doi:10.16288/j.yczz.17-011
Xu Liu, Ping Zhang, Xiaofeng Zhang, Xing Li, Yu Bai, Kerong Jia, Xiaodong Guo, Hao Zhang, Xiaoyan Ma, Ming Cang, Dongjun Liu, Xudong Guo.
成纤维细胞生长因子(fibroblast growth factors, FGFs)是一类小型肽类分子,在生长、发育、性别决定与分化等生物学过程中发挥重要作用[1,2]。研究发现,脊椎动物FGF家族包括7个亚家族,分别是FGF1/2、FGF4/5/6、FGF3/7/10/22、FGF8/17/18、FGF9/16/20、FGF11/12/13/14和FGF19/21/23。FGF家族成员多数需要与细胞表面的酪氨酸激酶FGF受体(fibroblast growth factor receptors, FGFRs)和肝素共结合后发挥其生物学功能[3]。但FGF19/FGF21/ FGF23亚家族缺少肝素结合区域,且需要与KLB (beta Klotho)结合形成共同复合体从而激活FGFR信号[4]。研究证明,FGF21的C末端、N末端与其生物活性密切相关：首先FGF21的C末端与KLB的跨膜蛋白结合,随后N末端与FGFR结合,形成稳定的FGF21/KLB/FGFR复合体,从而激活下游信号分子发挥其生物学效应[5,6]。小鼠(Mus musculus) Fgf21蛋白由210个氨基酸组成,氨基末端含有一段由30个氨基酸组成的典型信号肽序列[7]。Li等[8]研究发现,人FGF21编码基因与小鼠序列相似性约为75%。FGF21作为一种重要的调控因子,在脂肪、肝脏、脑和胰脏等组织的代谢调节、临床药理和生物免疫等方面发挥重要作用,但其作用机制还未被完全阐明。
目前,有关FGFs在毛囊生长发育及调控机理方面的研究越来越广泛。研究证明,凝集性生长的毛乳头细胞促进毛母质细胞增殖角化可能与FGF7有关[9];FGF5对毛囊生长发育、绒毛密度、绒毛长度有一定的调控作用[5];FGF10、FGF8和FGFR3的mRNA和蛋白水平在毛囊生长发育的不同部位与不同时期存在差异表达[10],表明它们可能与毛囊的生长发育具有一定的关系,并在不同水平发挥调控作用。此外,临床研究发现,FGF7与FGF2有可能参与了雄激素性脱发(androgenetic alopecia, AGA)[11],为进一步深入研究FGF7和FGF2对毛囊生长发育的调控作用提供了线索。这些研究结果为解析FGFs基因调控毛囊生长发育的分子机制奠定了基础,也为临床上毛囊再生研究与动物绒、毛候选基因的筛选提供了思路。FGF21作为FGF家族的一员,Dong等[12]通过生物信息学手段预测其对毛囊的生长发育及相关因子的调控也有重要的影响,但并未得到具体的实验证实。本研究利用CRISPR/Cas9技术构建了小鼠FGF21基因敲除载体,并快速建立了Fgf21-/-小鼠模型,为后续研究FGF21在毛囊发育及生长周期中的功能提供了更好的动物模型。
1 材料和方法
1.1 材料
本研究所用小鼠品系为FVB,该品系源自远交系Swiss鼠,是在美国NIH (National Institutes of Health, NIH)选育得到的Fv1b(易发B型白血病)同型合子近交系。动物实验符合内蒙古大学动物伦理委员会的规定并被授权。小鼠胎儿成纤维细胞和E. coli感受态细胞由本实验室保存。1.2 方法
1.2.1 gRNA设计和CRISPR/Cas9系统载体制备靶位点的选择是基因敲除的关键,通过序列的突变从而造成基因功能的缺失。FGF21基因位于7号染色体上,包含3个外显子。根据gRNA靶基因的设计原则,本研究在小鼠FGF21基因(Gene ID: 56636)第1外显子(185~422)设计靶位点,利用美国麻省理工学院CRISPR Design软件(http://crispr.mit.edu/)设计筛选得到4个gRNA,分别为：
FGF21-1(217~236)：5°-GCAGTCGGACCCACAGTCCCC-3°;
FGF21-2(306~325)：5°-TTGGGGGTCAAGTCCGGCA-3°;
FGF21-3(304~323)：5°-GCCGGACTTGACCCCCAAAC-3°;
FGF21-4(253~272)：5°-GATGCTTGGTAGACCCCCAG-3°。
将4个gRNA与对应的互补序列分别加上与BbsⅠ酶切CRISPR/Cas骨架质粒互补的粘性末端,合成对应的Oligo[13]。合成条件为：95℃ 3 min,缓慢冷却至25℃(-1℃/20 s),16℃ 20 min。将合成的gRNA单链复性形成的双链与线性化的CRISPR/Cas骨架进行连接,连接产物加入E. coli感受态细胞,经转化、涂板,并于37℃恒温倒置过夜。次日,挑取单菌落,进行菌液PCR,经琼脂糖凝胶电泳检测,确定为阳性细菌后进行测序。测序正确的样品继续摇菌,37℃、220 r/min恒温摇床培养过夜,提取质粒后分别命名为FGF21-gRNAN(N=1~4)和hCas9。
1.2.2 在细胞水平上利用Surveyor突变检测试剂盒检测FGF21敲除效率
将FGF21-gRNAN和hCas9质粒转染至汇合度达到60%~80% (8×106)的小鼠胎儿成纤维细胞,2 d后提取细胞基因组DNA。按照Surveyor突变检测试剂盒(日本TRANSGEMIC公司)说明书,分别加入1 μL Surveyor Nuclease S、1 μL Surveyor Enhancer S、 0.15 mol/L MgCl2 3 μL和20 μL DNA,混匀后42℃放置1 h,加入1/10体积的终止液混匀后用2%的琼脂糖凝胶进行电泳检测。
1.2.3 Cas9/gRNA体外转录
通过 XbaⅠ内切酶线性化hCas9质粒获得Cas9 mRNA体外转录模板,用mMESSAGE mMACHI- NE?T7 Ultra Kit(美国Life Technologies 公司)体外转录Cas9 mRNA, MEGAclear? Kit纯化回收Cas9 mRNA并进行电泳检测。以FGF21-F1和FGF21-R1为引物(引物序列见表1)扩增FGF21-gRNA质粒,得到 FGF21-gRNA体外转录模板。扩增程序：94℃预变性5 min;94℃变性30 s,56℃复性30 s,72℃延伸 50 s,共35个循环;最后再72℃延伸10 min,16℃保温。使用 MEGAshortscript? Kit体外转录FGF21- gRNA,使用MEGAclear? Kit纯化回收得到FGF21- gRNA并进行电泳检测。
Table 1
表1
表1 引物信息
Table 1
| 引物名称 | 引物序列(5°→3°) |
|---|---|
| FGF21-F1 | GTTCCTGCCAAGTGTGTCAAATA |
| FGF21-R1 | GGACCACAGAAAGAGACTCACCG |
| FGF21-F2 | TACTCCCATCTGGAGGACCC |
| FGF21-R2 | CACAAAGGTATGAGGGCGGA |
| gRNA-5°R | TGTACAAAAAAGCAGGCTTTAAAG |
| gRNA-3°F | CGGTGTTTCGTCCTTTCCACAAGA |
| gRNA-F | GTTTTAGAGCTAGAAATAGCAAG |
| gRNA-R | TAATGCCAACTTTGTACAAGAAAG |
| GAPDH-F | CACGATGGAGGGGCCGGACTCATC |
| GAPDH-R | TAAAGACCTCTATGCCAACACAGT |
新窗口打开|下载CSV
1.2.4 小鼠受精卵显微注射
根据文献[14,15]将体外转录好的FGF21-gRNA和Cas9 mRNA混合：取25 ng/μL FGF21-gRNA 20 μL和100 ng/μL Cas9 mRNA 10 μL混匀,加入3 mol/L乙酸钠3 μL,pH值调至5.2,用无水乙醇沉淀后再用无酶水重悬。通过显微注射法将混合物注射到小鼠受精卵胞质中,将存活的受精卵移植到假孕受体母鼠内,至母鼠生产获得首建鼠F0代。
1.2.5 基因型鉴定
受精卵移植的假孕母鼠单笼饲养20 d后,观察乳鼠出生情况。剪取长至1周后F0代小鼠尾尖组织1 cm,提取基因组DNA。在FGF21基因靶向区域使用FGF21-F1和FGF21-R1引物(引物序列见表1)进行PCR扩增,将PCR产物进行测序鉴定F0代小鼠基因型。
1.2.6 qRT-PCR和Western blotting验证
取野生型(WT)和Fgf21-/-小鼠的皮肤组织提取总RNA,经逆转录得到cDNA。利用引物FGF21-F2和FGF21-R2(引物序列见表1)进行qRT-PCR,以GAPDH为内参基因,检测FGF21 mRNA的表达情况。PCR扩增条件为：95℃ 5 min;95℃ 30 s,54℃ 30 s,72℃ 30 s,45个循环;72℃ 10 min,4℃保存。PCR产物用2%的琼脂糖凝胶电泳进行检测。
取WT和Fgf21-/-小鼠的背部皮肤组织,在含有蛋白酶抑制剂的匀浆中提取总蛋白,经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)分离。用含5%奶粉的磷酸缓冲液(phosphate buffer solution, PBS)的封闭液室温放置1 h,经FGF21抗体(ab644857,上海艾博抗贸易有限公司) 4℃孵育过夜,TBST洗3次,羊抗兔二抗(ab6721,上海艾博抗贸易有限公司)室温孵育1 h,TBST洗3次,经ECL显影液在化学发光仪中显影成像,检测FGF21蛋白表达情况。
1.2.7 FGF21组织表达谱和Fgf21-/-小鼠表型分析
取WT小鼠脑、心脏、肝脏、脾脏、肺、肾脏、胰脏、胸腺、胆囊、睾丸、卵巢、脂肪和肌肉组织各120 mg,分别剪碎后放于1.2 mL TRIzol (TaKaRa公司)中,用酚/氯仿抽提各种组织的RNA并溶于焦碳酸二乙酯(Diethylpyrocarbonate, DEPC)水中。以RNA为模板,根据PrimeScript? RT-PCR Kit说明书,用逆转录酶(大连TaKaRa公司)逆转录成cDNA,利用引物FGF21-F2和FGF21-R2(引物序列见表1)进行qRT-PCR,分析FGF21在WT小鼠各种组织中的表达情况。利用GAPDH(引物序列见表1)作为内参基因,使用??Ct方法计算FGF21的相对表达量,实验均重复3次。
定期观察Fgf21-/-小鼠的生长状况,取性别相同、8周龄的WT和Fgf21-/-小鼠称量体重并统计,分别取对照组(同窝WT小鼠)和Fgf21-/-小鼠各3只解剖观察比较器官组织生理情况。
1.2.8 人工脱毛诱导毛发进入生长期
小鼠的毛发生长周期约为17~19 d,包括生长期、退行期和静止期[2]。6~10周龄的小鼠毛发均已进入静止期,通过人工脱毛处理可诱导小鼠毛发从静止期进入生长期[3]。待WT和Fgf21-/-小鼠长至6~10周龄后,将蜡和松香按1∶1混合加热至融化后,用玻璃棒涂抹于小鼠背部皮肤,待混合物凝固后,用镊子逆着小鼠毛发生长的方向脱毛处理,实时监测毛发再生速度。
1.2.9 石蜡切片及染色
取小鼠脱毛后长毛程度相同的WT和Fgf21-/-小鼠皮肤组织,经4%多聚甲醛固定24 h,流水冲洗24 h,无水乙醇脱水3 h,二甲苯透明1 h处理后用石蜡包埋,并用石蜡切片机(德国Leica公司)切成 6 μm厚的组织切片,进行H&E(hematoxylin-eosin staining)染色,用中性树胶封片,显微镜观察。
2 结果与分析
2.1 gRNA表达载体的构建
2.1.1 gRNA片段扩增结果利用引物gRNA-F和gRNA-R、gRNA-5′R和gRNA-3′F(表1)扩增gRNA、gRNA 5′端和 gRNA 3′端,用2%的琼脂糖凝胶电泳检测,得到gRNA目的条带与设计的gRNA一致,表明gRNA载体构建成功(图1A)。
图1
新窗口打开|下载原图ZIP|生成PPT图1gRNA的鉴定及Cas9 mRNA和FGF21-gRNA的体外转录
A：gRNA PCR扩增产物电泳图。M：DL1000 marker; 1：gRNA 5′端(319 bp);2：gRNA 3′端(177 bp);3：gRNA(455 bp)。B：Surveyor酶检测FGF21敲除效率。M：100 bp marker;Mock：未处理的细胞;1、2、3、4分别为转染FGF21-gRNAN(N=1~4)后的结果;红色箭头代表切割的预期大小,表示基因发生突变。C：Cas9 mRNA和FGF21-gRNA体外转录结果。M：DL2000 marker;1~3：分别为FGF21-gRNA1、FGF21-gRNA2和Cas9 mRNA。
Fig. 1Identification of gRNA and in vitro transcription of Cas9 mRNA and FGF21-gRNA
2.1.2 细胞水平检测FGF21敲除效率
按照Surveyor突变检测试剂盒说明书,将细胞提取DNA与Surveyor试剂形成的反应液混匀后,
在2%的琼脂糖凝胶电泳检测,结果显示gRNA1和gRNA2敲除效率较高且无拖尾现象(图1B),因此选用gRNA1和gRNA2作为实验靶位点gRNA。
2.1.3 FGF21-gRNA和 Cas9 mRNA体外转录
利用构建好的gRNA1和gRNA2质粒制备相应的体外转录产物,经体外转录得到的gRNA浓度均在500 ng/μL以上,电泳结果表明其条带完整(图1C)。得到的Cas9 mRNA的浓度为100 ng/μL,电泳条带存在拖带情况(图1C),这可能是因为Cas9 mRNA基因序列长度超出试剂盒最佳转录范围,导致体外转录时Cas9 mRNA存在部分序列转录不完全。但从图中可以发现,Cas9 mRNA主带清晰,说明转录产物完整。
2.2 Fgf21-/-小鼠基因型鉴定
将转录的FGF21-gRNA和Cas9 mRNA 混合物注射到340颗受精卵,挑选状态良好的322颗受精卵移植到16只假孕FVB母鼠体内,约3周后共获得63只F0代小鼠,出生率为19.6%(表2)。提取F0代小鼠基因组DNA,利用引物FGF21-F2和FGF21- R2(引物序列见表1)进行PCR扩增,PCR产物送测序。经测序分析与WT小鼠基因组序列进行比对后,对F0代小鼠进行FGF21进行敲除情况统计(表2)。在获得的18只FGF21基因敲除小鼠中,共有3只Fgf21-/-小鼠。Fgf21-/-小鼠原始测序序列比对与峰图见图2。Table 2
表2
表2 显微注射获得FGF21基因敲除小鼠
Table 2
| 注射 卵数 | 移植 卵数 | F0出生小鼠 (出生率) | 首建鼠 (突变率) | 双敲小鼠 (突变率) | 单敲小鼠 (突变率) |
|---|---|---|---|---|---|
| 340 | 322 | 63(19.6%) | 18(28.6%) | 3(4.8%) | 15(23.8%) |
新窗口打开|下载CSV
图2
新窗口打开|下载原图ZIP|生成PPT图2Fgf21-/-小鼠基因型鉴定
A：Fgf21-/-(-103 bp)小鼠;B：Fgf21-/-(-109 bp)小鼠;C：Fgf21-/-(-17 bp)小鼠。黄色区表示第1个靶位点,绿色区表示第2个靶位点,红色虚线表示缺失基因。
Fig. 2Genotype identification of Fgf21-/- mice
2.3 qRT-PCR和Western blotting鉴定Fgf21-/-小鼠FGF21 mRNA和FGF21蛋白表达
利用qRT-PCR和Western blotting检测WT和Fgf21-/-小鼠背部皮肤中FGF21基因及蛋白表达情况。qRT-PCR结果表明,与WT相比,Fgf21-/-小鼠背部皮肤中未检测到FGF21 mRNA表达(图3A)。以α-tubulin为内参,通过Western blotting检测FGF21蛋白表达情况(图3B)。结果表明,在Fgf21-/-小鼠的背部皮肤中未检测到大小为22 kDa的FGF21蛋白表达,表明Fgf21-/-小鼠构建成功,可以用于后续分析。图3
新窗口打开|下载原图ZIP|生成PPT图3Fgf21-/-小鼠FGF21 mRNA和FGF21蛋白表达情况
A：qRT-PCR检测FGF21 mRNA表达。M：DL500 bp;WT：野生型;KO：Fgf21-/-小鼠。B：Western blotting检测FGF21蛋白表达。WT：野生型小鼠;KO1、KO2、KO3：Fgf21-/-小鼠。
Fig. 3FGF21 mRNA and FGF21 protein expression of Fgf21-/- by qRT-PCR and Western blotting
2.4 FGF21组织表达谱和Fgf21-/-小鼠表型分析
2.4.1 FGF21基因在WT小鼠各组织中的表达为研究Fgf21-/-小鼠的表型及敲除FGF21基因后的影响,本文首先利用qRT-PCR检测FGF21基因在WT小鼠各组织中的mRNA表达水平。结果表明,FGF21基因在胰脏中高表达,在脂肪、肝脏、睾丸、肌肉、胸腺、皮肤、脑、肺、卵巢、心脏、肾脏、胃、脾脏、胆囊等组织中表达水平较低(图4),结果与Fon等[16]的研究结果相一致。
图4
新窗口打开|下载原图ZIP|生成PPT图4FGF21基因组织表达谱
Fig. 4FGF21 mRNA expression in tissues
2.4.2 Fgf21-/-小鼠表型分析
通过对出生的Fgf21-/-和WT小鼠的观察比较,未发现异常变化,生长状况良好。对8周龄的WT和Fgf21-/-小鼠称重统计发现,WT雄性小鼠平均体重为24.52 g,雌性为23.28 g;Fgf21-/-小鼠雄性平均体重为24.35 g,雌性为23.22 g。结果证明,处于同周龄同性别的Fgf21-/-小鼠体重明显轻于WT小鼠,这可能与FGF21基因对肌肉脂肪组织的调控有关[4]。通过解剖学初步发现,与WT小鼠相比,Fgf21-/-小鼠各组织形态颜色没有明显差别,并且无组织病变状况出现,尤其是FGF21基因高表达的胰脏(图5),而FGF21敲除后对小鼠的组织生理影响还需进一步的研究。
图5
新窗口打开|下载原图ZIP|生成PPT图5WT和Fgf21-/-小鼠解剖分析
A：WT小鼠解剖图(左上放大图为胰腺);B：Fgf21-/-小鼠解剖图(左上放大图为胰腺)。
Fig. 5The anatomical analyses of WT and Fgf21-/- mice
2.5 FGF21基因对小鼠毛发影响
通过观察WT小鼠和3只Fgf21-/-小鼠皮肤毛发生长变化,发现脱毛后第12 d,WT小鼠和3只Fgf21-/-小鼠毛发生长差异显著。野生小鼠已长出较长、较厚的被毛(图6A);3只Fgf21-/-小鼠皮肤颜色只是变白,体表基本没有长出毛发(图6：B~D)。结果证明,与WT小鼠相比,Fgf21-/-小鼠毛发生长速度相对较慢。选取脱毛后毛发长势相同的皮肤组织,进行组织切片实验(WT：12 d;Fgf21-/-：17 d),观察处于毛发生长程度一致的小鼠毛囊变化。结果发现,与WT小鼠相比(图6A),Fgf21-/-小鼠的毛囊数量减少,并且毛囊直径变小(图6：B~D)。
图6
新窗口打开|下载原图ZIP|生成PPT图6WT和Fgf21-/-小鼠毛发再生速度的比较和组织切片观察
A：WT小鼠毛发再生情况及组织切片观察;B~D：Fgf21-/-小鼠毛发再生情况及组织切片观察。红色框区为一个毛囊;WT：野生型小鼠;KO：Fgf21-/-小鼠。
Fig. 6The comparison of the hair re-growth speed and paraffin sectionin WT and Fgf21-/- mice
3 讨 论
2010年后,制备遗传基因修饰动物模型的方法主要依赖于锌指核酸酶(zinc finger nucleases, ZFN) 技术、转录激活因子样效应核酸酶(transcription activator-like effector nucleases, TALENs)技术及CRISPR/Cas9技术[17]。相比ZFN和TALENs技术,CRISPR/Cas9系统具有设计简单、操作方便、费用低廉等显著优势[18,19],给生命科学研究的各个领域带来了革命性的改变。该技术只需要依据目的基因DNA的序列设计一段与之互补的gRNA序列,在其介导下可快速实现对基因组DNA的打靶,并且其打靶效率高,方法操作简单,实验周期短,可以在细胞内同时进行多个基因编辑操作[20],对生物学领域的发展起到深远影响。本研究利用CRISPR/Cas9系统,通过显微操作构建FGF21基因敲除小鼠模型,但结果只得到3只Fgf21-/-小鼠,敲除效率为28.6%,造成这种情况的原因主要是显微注射技术操作难度大。此外,DNA纯化回收、体外转录过程、注射片段FGF21-gRNA 和Cas9 mRNA浓度的调整都是非常重要的因素。本研究初步建立CRISPR/Cas9介导的基因敲除小鼠模型的技术体系,但是多个环节都有待改进。
FGF21以自分泌、旁分泌和内分泌的形式作用于多种靶器官内并调节体内内环境的能量平衡。由于FGF21的生理复杂性和不同的代谢功能,对肝脏、脂肪、胰腺等组织的脂肪酸和葡萄糖代谢等有重要的调控作用[21],而且能降低血糖、甘油三酯、果糖胺以及高血糖素浓度,改善胰岛β细胞功能,有望成为治疗肥胖症、糖尿病和脂肪肝等疾病的新一代“药物”。同时,FGF21也是最新发现的一个影响毛囊周期和结构的基因,通过生物信息学研究发现其功能可能是在毛囊发育中促进转变成退行期[12],推测FGF21在毛囊发育和周期中发挥着重要的作用,但并没有具体实验验证。因为FGF21在毛囊发育调控的机制研究还处于相对粗浅的水平,本实验通过构建CRISPR/Cas9表达载体及制备FGF21基因敲除小鼠,发现Fgf21-/-小鼠毛发生长速度明显减慢,毛囊直径变小,从而间接推断FGF21在毛囊发育过程中发挥重要作用,对阐明FGF21在毛囊周期中的调控作用提供实验依据。本研究利用CRISPR/Cas9技术成功构建了FGF21基因敲除小鼠模型,为今后进一步探究FGF21的结构、功能、靶基因以及对毛囊发育的作用及生长周期的调控机制提供参考。
参考文献 原文顺序
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被引期刊影响因子
[本文引用: 1]
URLPMID:3986619 [本文引用: 2]
Abstract Since the discovery of insulin in 1921, protein therapeutics have become vital tools in the treatment of diabetes mellitus. This heritage has been extended with the comparatively recent introduction of recombinant and re-engineered insulins, in addition to the advent of GLP1 agonists. FGF21 represents an example of a novel experimental protein therapy which is able to induce favorable metabolic effects in various species ranging from rodents to man. The aim of this review is to communicate the story of the FGF21 drug discovery path from identification in a functional in vitro screen, to the eventual evaluation of its utility in patients. Given that the development of FGF21 advanced hand-in-hand with rapidly evolving scientific research around this target, we have also attempted to describe our view of recent developments regarding the mechanistic understanding of FGF21 biology.
[本文引用: 2]
URLPMID:3612009 [本文引用: 2]
Abstract Dietary intake of macronutrients (carbohydrate, protein, and fat) has been associated with risk of chronic conditions such as obesity and diabetes. Family studies have reported a moderate contribution of genetics to variation in macronutrient intake. In a genome-wide meta-analysis of a population-based discovery cohort (n = 33 533), rs838133 in FGF21 (19q13.33), rs197273 near TRAF family member-associated NF-kappa-B activator (TANK) (2p24.2), and rs10163409 in FTO (16q12.2) were among the top associations (P < 10(-5)) for percentage of total caloric intake from protein and carbohydrate. rs838133 was replicated in silico in an independent sample from the Cohorts for Heart and Aging Research in Genomic Epidemiology Consortium (CHARGE) Nutrition Working Group (n = 38 360) and attained genome-wide significance in combined analysis (Pjoint = 7.9 脙聴 10(-9)). A cytokine involved in cellular metabolism, FGF21 is a potential susceptibility gene for obesity and type 2 diabetes. Our results highlight the potential of genetic variation for determining dietary macronutrient intake.
URLPMID:10858549 [本文引用: 2]
We isolated cDNA encoding a novel FGF (210 amino acids) from mouse embryos. As this is the 21st documented FGF, we tentatively term it FGF-21. FGF-21 has a hydrophobic amino terminus (6530 amino acids), which is a typical signal sequence, and appears to be a secreted protein. The expression of FGF-21 mRNA in mouse adult tissues was examined by Northern blotting analysis. FGF-21 mRNA was most abundantly expressed in the liver, and also expressed in the thymus at lower levels. We also isolated human cDNA encoding FGF-21 (209 amino acids). Human FGF-21 is highly identical (6575% amino acid identity) to mouse FGF-21. Among human FGF family members, FGF-21 is most similar (6535% amino acid identity) to FGF-19.
URLPMID:23150685 [本文引用: 1]
Abstract CONTEXT: Cold exposure stimulates fibroblast growth factor 21 (FGF21) secretion in animals, enhancing the cold-induced thermogenesis (CIT) response through browning of white adipose tissue. In humans, the effects of cold exposure on circulating FGF21 levels are unknown. OBJECTIVE: Our objective was to evaluate the effects of mild cold exposure on circulating FGF21 and its relationship with CIT and lipolysis in humans. DESIGN AND SETTING: We conducted a randomized, single-blind, crossover intervention study at the National Institutes of Health Clinical Center. PARTICIPANTS: Participants were healthy adults. INTERVENTION: Subjects were exposed to a 12-h exposure to 24 or 19 C in a whole-room indirect calorimeter. OUTCOME MEASURES: Energy expenditure, plasma FGF 21, nonesterified fatty acid, and adipose tissue microdialysis glycerol concentrations were evaluated. RESULTS: At 24 C, plasma FGF21 exhibited a diurnal rhythm, peaking at 0800 h [110 (59-178) pg/ml], and progressively dropped to a nadir at 1700 h [41 (21-71) pg/ml, P < 0.0001] before rising at 1900 h [60 (11-81) pg/ml, P < 0.0001]. Exposure at 19 C lessened the diurnal reduction of FGF21 observed at 24 C from 0800-1700 h and augmented overall FGF21 levels by 37 卤 45% (P = 0.01). The change in area under the curve plasma FGF21 between 19 and 24 C correlated positively with the change in area under the curve adipose microdialysate glycerol (R(2) = 0.35, P = 0.04) but not with nonesterified fatty acid. Cold-induced increase in FGF21 predicted greater rise in energy expenditure during cold exposure (尾 = 0.66, P = 0.027), independent of age, gender, fat mass, and lean mass. CONCLUSIONS: Mild cold exposure increased circulating FGF21 levels, predicting greater lipolysis and CIT. A small reduction in environmental temperature is sufficient to modulate FGF21 diurnal rhythm in humans, which may mediate cold-induced metabolic changes similar to those in animals.
URLPMID:24049735 [本文引用: 1]
Fibroblast growth factor 21 is an emerging metabolic regulator that was recently proposed to be a fed-state inducible factor in adipose tissue. As mice lacking FGF21 were refractory to treatment with rosiglitazone, FGF21 was suggested to underlie PPARγ-driven pharmacology and side effect profile (Dutchak et al., 2012 [12]). To evaluate FGF21/PPARγ cross-talk we conducted experiments in control and FGF21 null animals and found that rosiglitazone was equally efficacious in both strains. Specifically, diverse endpoints ranging from enhanced glycemic control, improved lipid homeostasis and side effects such as adipose accumulation were evident in both genotypes. Furthermore, the transcriptional signature and cytokine secretion profile of rosiglitazone action were maintained in our FGF21KO animals. Finally, we found that FGF21 in adipose was expressed at comparable levels in fasted and fed states. Thus, our data present a new viewpoint on the FGF21/PPARγ interplay whereby FGF21 is not necessary for the metabolic events downstream of PPARγ.
URLPMID:22455565 [本文引用: 1]
Abstract Recently, autologous platelet-rich plasma (PRP) has attracted attention in various medical fields, including plastic and orthopedic surgery and dermatology, for its ability to promote wound healing. PRP has been tested during facelift and hair transplantation to reduce swelling and pain and to increase hair density. To investigate the effects of PRP on hair growth using in vivo and in vitro models. PRP was prepared using the double-spin method and applied to dermal papilla (DP) cells. The proliferative effect of activated PRP on DP cells was measured. To understand the mechanisms of activated PRP on hair growth, we evaluated signaling pathways. In an in vivo study, mice received subcutaneous injections of activated PRP, and their results were compared with control mice. Activated PRP increased the proliferation of DP cells and stimulated extracellular signal-regulated kinase (ERK) and Akt signaling. Fibroblast growth factor 7 (FGF-7) and beta-catenin, which are potent stimuli for hair growth, were upregulated in DP cells. The injection of mice with activated PRP induced faster telogen-to-anagen transition than was seen on control mice. Although few studies tested the effects of activated PRP on hair growth, this research provides support for possible clinical application of autologous PRP and its secretory factors for promotion of hair growth.
URLPMID:4728323 [本文引用: 1]
MAP3K1 is a serine/threonine kinase that is activated by a diverse set of stimuli and exerts its effect through various downstream effecter molecules, including JNK, ERK1/2 and p38. In humans, mutant alleles of MAP3K1 are associated with 46,XY sex reversal. Until recently, the only phenotype observed in Map3k1(tm1Yxia) mutant mice was open eyelids at birth. Here, we report that homozygous Map3k1(tm1Yxia) mice have early-onset profound hearing loss accompanied by the progressive degeneration of cochlear outer hair cells. In the mouse inner ear, MAP3K1 has punctate localization at the apical surface of the supporting cells in close proximity to basal bodies. Although the cytoarchitecture, neuronal wiring and synaptic junctions in the organ of Corti are grossly preserved, Map3k1(tm1Yxia) mutant mice have supernumerary functional outer hair cells (OHCs) and Deiters' cells. Loss of MAP3K1 function resulted in the downregulation of Fgfr3, Fgf8, Fgf10 and Atf3 expression in the inner ear. Fgfr3, Fgf8 and Fgf10 have a role in induction of the otic placode or in otic epithelium development in mice, and their functional deficits cause defects in cochlear morphogenesis and hearing loss. Our studies suggest that MAP3K1 has an essential role in the regulation of these key cochlear morphogenesis genes. Collectively, our data highlight the crucial role of MAP3K1 in the development and function of the mouse inner ear and hearing.
URLPMID:27040904 [本文引用: 1]
Abstract Alopecia is an important issue that can occur in people of all ages. Recent studies show that bee venom can be used to treat certain diseases including rheumatoid arthritis, neuralgia, and multiple sclerosis. In this study, we investigated the preventive effect of bee venom on alopecia, which was measured by applying bee venom (0.001, 0.005, and 0.01%) or minoxidil (2%) as a positive control to the dorsal skin of female C57BL/6 mice for 19 days. Growth factors responsible for hair growth were analyzed by quantitative real-time PCR and western blot analysis using mice skins and human dermal papilla cells (hDPCs). Bee venom promoted hair growth and inhibited transition from the anagen to catagen phase. In both anagen phase mice and dexamethasone-induced catagen phase mice, hair growth was increased dose dependently compared with controls. Bee venom inhibited the expression of SRD5A2, which encodes a type II 5伪-reductase that plays a major role in the conversion of testosterone into dihydrotestosterone. Moreover, bee venom stimulated proliferation of hDPCs and several growth factors (IGF-1R, VEGF, FGF2, and FGF7) in bee venom-treated hDPCs dose dependently compared with the control group. In conclusion, bee venom is a potentially potent 5伪-reductase inhibitor and hair growth promoter.
URLPMID:5068700 [本文引用: 1]
Abstract Precision genetic engineering accelerates the genetic improvement of livestock for agriculture and biomedicine. We have recently reported our success in producing gene-modified goats using the CRISPR/Cas9 system through microinjection of Cas9 mRNA and sgRNAs targeting the MSTN and FGF5 genes in goat embryos. By investigating the influence of gene modification on the phenotypes of Cas9-mediated goats, we herein demonstrate that the utility of this approach involving the disruption of FGF5 results in increased number of second hair follicles and enhanced fiber length in Cas9-mediated goats, suggesting more cashmere will be produced. The effects of genome modifications were characterized using H&E and immunohistochemistry staining, quantitative PCR, and western blotting techniques. These results indicated that the gene modifications induced by the disruption of FGF5 had occurred at the morphological and genetic levels. We further show that the knockout alleles were likely capable of germline transmission, which is essential for goat population expansion. These results provide sufficient evidences of the merit of using the CRISPR/Cas9 approach for the generation of gene-modified goats displaying the corresponding mutant phenotypes.
URL [本文引用: 2]
URL [本文引用: 1]
体细胞核移植技术在家畜良种繁育、基因修饰动物生产、濒危动物的拯救和人类疾病的治疗等领域具有重要的应用价值，但目前克隆动物生产效率较低，平均不超过5%。低下的克隆效率极大地限制了该技术的快速发展。在影响克隆猪生产效率的诸多因素中，X染色体的异常失活是导致克隆效率低下的重要原因，而与X染色体失活密切相关的一个重要基因是Xist基因，这表明Xist基因可能直接或间接地影响猪的克隆效率。本文以CRISPR/Cas系统为基础，在Xist基因上设计5个CRISPR/Cas系统打靶位点，并筛选出有效的Target 3、Target 4 sgRNA，在细胞水平切割效率为1%和3%，在胚胎水平为75%和85.7%。同时将有效的sgRNA体外转录并显微注射至胚胎体内，发现Target 3和Target 4组合效果最好，敲除效率为100%。通过胞浆注射和胚胎移植方法生产出6头克隆猪，有2头活仔实现完全敲除。本文成功建立Xist基因敲除猪模型，为后续通过敲除猪Xist基因提高克隆效率的研究奠定了基础。
URL [本文引用: 1]
体细胞核移植技术在家畜良种繁育、基因修饰动物生产、濒危动物的拯救和人类疾病的治疗等领域具有重要的应用价值，但目前克隆动物生产效率较低，平均不超过5%。低下的克隆效率极大地限制了该技术的快速发展。在影响克隆猪生产效率的诸多因素中，X染色体的异常失活是导致克隆效率低下的重要原因，而与X染色体失活密切相关的一个重要基因是Xist基因，这表明Xist基因可能直接或间接地影响猪的克隆效率。本文以CRISPR/Cas系统为基础，在Xist基因上设计5个CRISPR/Cas系统打靶位点，并筛选出有效的Target 3、Target 4 sgRNA，在细胞水平切割效率为1%和3%，在胚胎水平为75%和85.7%。同时将有效的sgRNA体外转录并显微注射至胚胎体内，发现Target 3和Target 4组合效果最好，敲除效率为100%。通过胞浆注射和胚胎移植方法生产出6头克隆猪，有2头活仔实现完全敲除。本文成功建立Xist基因敲除猪模型，为后续通过敲除猪Xist基因提高克隆效率的研究奠定了基础。
[本文引用: 1]
URLPMID:24002648 [本文引用: 1]
Abstract We report a simple yet extremely efficient platform for systematic gene targeting by the RNA-guided endonuclease Cas9 in Drosophila. The system comprises two transgenic strains: one expressing Cas9 protein from the germline-specific nanos promoter and the other ubiquitously expressing a custom guide RNA (gRNA) that targets a unique site in the genome. The two strains are crossed to form an active Cas9-gRNA complex specifically in germ cells, which cleaves and mutates the target site. We demonstrate rapid generation of mutants in seven neuropeptide and two microRNA genes in which no mutants have been described. Founder animals stably expressing Cas9-gRNA transmitted germline mutations to an average of 60% of their progeny, a dramatic improvement in efficiency over the previous methods based on transient Cas9 expression. Simultaneous cleavage of two sites by co-expression of two gRNAs efficiently induced internal deletion with frequencies of 4.3-23%. Our method is readily scalable to high-throughput gene targeting, thereby accelerating comprehensive functional annotation of the Drosophila genome.
URL [本文引用: 1]
URLPMID:4230364 [本文引用: 1]
The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) is a simple, robust and efficient technique for gene targeting in model organisms such as zebrafish, mice and rats. In this report, we applied CRISPR technology to rabbits by microinjection of Cas9 mRNA and guided RNA (gRNA) into the cytoplasm of pronuclear-stage embryos. We achieved biallelic gene knockout (KO) rabbits by injection of 1 gene (IL2rg) or 2 gene (IL2rg and RAG1) Cas9 mRNA and gRNA with an efficiency of 100%. We also tested the efficiency of multiple gene KOs in early rabbit embryos and found that the efficiency of simultaneous gene mutation on target sites is as high as 100% for 3 genes (IL2rg, RAG1 and RAG2) and 33.3% for 5 genes (IL2rg, RAG1, RAG2, TIKI1 and ALB). Our results demonstrate that the Cas9/gRNA system is a highly efficient and fast tool not only for single-gene editing but also for multi-gene editing in rabbits.
URLPMID:25430965 [本文引用: 1]
The article focuses on the use of site-specific nucleases such as Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) for carrying out genome modification in non-human primates (NHPs). It states that p53 tumor suppressor gene, injected via a zygote injection using a CRISPR system, was used to produce biallelic mutant monkeys. It states that addition of desired template DNA to the CRISPR system can modify nucleotide.
URLPMID:23992847 [本文引用: 1]
The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.
URLPMID:24076990 [本文引用: 1]
Abstract RNA-guided Cas9 nucleases derived from clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have dramatically transformed our ability to edit the genomes of diverse organisms. We believe tools and techniques based on Cas9, a single unifying factor capable of colocalizing RNA, DNA and protein, will grant unprecedented control over cellular organization, regulation and behavior. Here we describe the Cas9 targeting methodology, detail current and prospective engineering advances and suggest potential applications ranging from basic science to the clinic.
URLPMID:26654352 [本文引用: 1]
Fibroblast growth factor 21 (FGF21) is a peptide hormone that is synthesized by several organs and regulates energy homeostasis. Excitement surrounding this relatively recently identified hormone is based on the documented metabolic beneficial effects of FGF21, which include weight loss and improved glycemia. The biology of FGF21 is intrinsically complicated owing to its diverse metabolic functions in multiple target organs and its ability to act as an autocrine, paracrine, and endocrine factor. In the liver, FGF21 plays an important role in the regulation of fatty acid oxidation both in the fasted state and in mice consuming a high-fat, low-carbohydrate ketogenic diet. FGF21 also regulates fatty acid metabolism in mice consuming a diet that promotes hepatic lipotoxicity. In white adipose tissue (WAT), FGF21 regulates aspects of glucose metabolism, and in susceptible WAT depots, it can cause browning. This peptide is highly expressed in the pancreas, where it appears to play an anti-inflammatory role in experimental pancreatitis. It also has an anti-inflammatory role in cardiac muscle. Although typically not expressed in skeletal muscle, FGF21 is induced in situations of muscle stress, particularly mitochondrial myopathies. FGF21 has been proposed as a novel therapeutic for metabolic complications such as diabetes and fatty liver disease. This review aims to interpret and delineate the ever-expanding complexity of FGF21 physiology. Expected final online publication date for the Annual Review of Physiology Volume 78 is February 10, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
