Generation of Foxo3‐targeted Mice by Injection of mRNAs ...Ÿº因敲除小鼠... · Generation of Foxo3-targeted Mice by Injection of mRNAs Encoding Transcription Activator-like

Generation of Foxo3-targeted Mice by Injection of mRNAs Encoding Transcription

Activator-like Effector Nucleases (TALENs) into Zygotes

P Zhu1,*, Q Liu1,*, S Liu1, X Su1, W Feng1, X Lei1, J Liu1, K Cui1, B Huang1,2 and D Shi1

1State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, Guangxi, China; 2GuangxiExperiment Centre of Science and Technology, Guangxi University, Nanning, Guangxi, China

ContentsIn this study, for exploring the mechanism of forkhead box O3(Foxo3) participating in regulation of the activation ofprimordial oocytes, Foxo3-targeted mice were generated byinjection of mRNAs encoding transcription activator-likeeffector nucleases (TALENs) into mouse zygotes. The TALENsites were designed with high conservative homologous regionamong pig, bovine, buffalo and mouse by commercial bio-companies. The TALENs mutagenic non-homologous end-joining (NHEJ) repair activity were determined to be 31.3% inhuman embryonic kidney 293T (HEK-293T) cells by dualluciferase reporter assay system. Then, we firstly injectedTALEN-mRNAs into the cytoplasm of mouse zygotes bymicromanipulation, and four of 48 mouse blastocysts wereidentified as mutation by sequencing. Subsequently, by themethod of TALEN-mRNAs injected into the zygotes withpronucleus micromanipulation technique, we obtained sevenFoxo3 mutants of 20 FVB/NJ backgrounds mice which wereFoxo3-independent alleles with frameshift and deletion muta-tions. It was very interesting that all seven were heterozygousmutants (Foxo3�/+), and the gene mutagenesis rates of themice reached 35%. The five Foxo3 mutant females were allinfertile in the following 6 months after birth. The histologicalexamination results showed that there were rare primordialfollicles and primary follicles in the ovary of Foxo3 mutantcompared to that of wide-type female mice. Moreover, one oftwo mutant males was subfertile and another was fertilenormally. Those results suggested that the mutant of Foxo3severely affected the fertile ability of female and perhaps malein some degree; furthermore, an even more efficient TALENs-based gene mutation method has been established to be poisedto revolutionize the study of mouse and other species genetics.

Introduction

The ability to precisely modify the mouse genomeexperimentally has had a considerable impact over thelast 25 years in diverse areas of biomedical research andhas made the mouse one of the most important modelorganisms in the laboratory today. Alterations in thegenome are conventionally made by the process of genetargeting in embryonic stem cell via homologousrecombination (Doyle et al. 2012). Using homologousrecombination, whole genes or exons can be deletedfrom the mouse genome and the phenotypic conse-quences of these knockout models can deliver importantinformation concerning gene function. However, the

generation of knockout animals via the traditionalmethod, such as homologous recombination, is a time-consuming and laborious process, and therefore, amore-efficient tool is preferred for generating knockoutmouse. Targeted gene knockdown by RNAi has pro-vided researchers with a rapid, inexpensive and high-throughput alternative to homologous recombination(McManus and Sharp 2002). Yet, knockdown by RNAiis incomplete, varies between experiments and labora-tories, has unpredictable off-target effects and providestemporary inhibition of gene function or can bepermanently knockdown using transgenic approachin vivo or lentiviral vectors in vitro.In the past decade, a new approach has emerged that

enables investigators to manipulate virtually any gene ina diverse range of cell types and organisms. Theversatility of this approach is facilitated by the pro-grammability of the DNA-binding domains that arederived from zinc-finger and transcription activator-likeeffector (TALE) proteins. This combination of simplic-ity and flexibility has catapulted zinc-finger nucleases(ZFNs) (Urnov et al. 2010; Carroll 2011) and transcrip-tion activator-like effector nucleases (TALENs)( Ikmiet al. 2014; Moghaddassi et al. 2014; Sun et al. 2014).Also, in the past years, a new approach has beendeveloped and applied, it is just the clustered regulatoryinterspaced short palindromic repeats CRISPR/Cas(CRISPR-associated)-based RNA-guided DNA endo-nucleases (Cho et al. 2013; Cong et al. 2013; Mali et al.2013). Up to now, these engineered TAL effectornucleases (TALENs) have been successfully applied todisrupt gene function in the yeast (Li et al. 2011), rice(Li et al. 2014), zebrasfish (Tatsumi et al. 2014), bovine(Yang et al. 2014), mouse (Nakagawa et al. 2014),human (Fadel et al. 2014) and so on. Knocking-outgenes contain both protein coding genes (Bradley et al.2012) and microRNA (Prosser et al. 2011; Zhang et al.2013).Foxo3 is a member of the FOXO (forkhead box class

O) transcription factors family and consists of fourexons and produce a protein of a 673 amino acids. It hasroles in cell cycle arrest (Medema et al. 2000; Alvarezet al. 2001), apoptosis (Dijkers et al. 2000; Modur et al.2002) and DNA repair (Tran et al. 2002; Luo et al.2006) among other functions. Consistent with theirability to block cell growth, inactivation of FOXOproteins is thought to be a critical event in oncogenic

*Those authors contributed to this article equivalently.

Reprod Dom Anim 50, 474–483 (2015); doi: 10.1111/rda.12515

ISSN 0936–6768

© 2015 Blackwell Verlag GmbH

transformation (Greer and Brunet 2005; Fu and Tindall2008). Deletion of all FOXO1, Foxo3 and FOXO4alleles in adult mice induced a cancer prone phenotype,supporting a tumour-suppressing function of theseproteins (Paik et al. 2007). In addition to their role incell cycle regulation, FOXO transcription factors alsoregulate glucose metabolism in various organs (Mat-sumoto et al. 2007; Haeusler et al. 2010) and increasethe resistance to oxidative stress (Kops et al. 2002;Brunet et al. 2004). Furthermore, the FOXO orthologueDAF-16 controls lifespan extension in Caenorhabditiselegans together with homologues of the human insulinreceptor and the phosphatidylinositol 3-kinase (DAF-2and AGE-1) (Lin et al. 1997; Ogg et al. 1997). Specif-ically Foxo3�/� female mice had a very singularphenotype which they were initially fertile and subse-quently infertile after 15 weeks of age (Castrillon et al.2003; Hosaka et al. 2004). The activation of primordialfollicle was presented in Foxo3 knockdown pigs (Mon-iruzzaman et al. 2010), but the mechanism remains to befurther clarified.In this study, we were keen to produce Foxo3

knockdown mice by TALEN-based targeting techniqueand, consequently, able to establish an efficient gene-targeting system for creating heritable mutations toapply for the study of mouse and other species genetics.

Materials and methods

TALENs and reporters

Plasmids encoding the TALENs pCS2-peas-T andreporter pSSA-LUC used in this study were obtainedfrom viewsolid biotech (Beijing, China). Reporter pRGSwere prepared as previously described (Kim et al. 2011).Briefly, oligonucleotides that contained target sites weresynthesized (Sangon biotech, Shanghai, China) andannealed in vitro. The annealed oligonucleotides wereligated into the pRGS and pSSA-LUC (Table 1). AfterDNA for each of the Foxo3-TALEN and reporterplasmids were prepared using the Qiagen Endo-freeMaxi-prep kit as directed by the manufacturer, reporterplasmid co-transfection of the Foxo3-TALEN vectorsinto HEK293T cells led to nuclease-dependent generepair and enhanced green fluorescent protein (eGFP)and luciferase were expressed.

Cell culture and transfection

HEK293T cells were maintained in Dulbecco’s modifiedEagle medium (DMEM, Invitrogen) supplemented with100 units/ml penicillin, 100 mg/ml streptomycin, and10% foetal bovine serum. Cells were transfected usinglipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) ata weight ratio of 1 : 1 : 2 (plasmid encoding a TALEN:plasmid encoding the other TALEN: pRGS reporter) or100 ng plasmid encoding a TALEN: 100 ng plasmidencoding the other TALEN: 50 ng pSSA-LUC reporter:10 ng pRL-TK (Promega, Beijing, China).

Dual luciferase reporter assay

The 293T cells were seeded in 24-well plates. After 24-hincubation, cells were transfected with TALENs andpSSA-Luc (Sajwan et al. 2013; Aryan et al. 2013). Forty-eight hours after transfection, the cells were assayed byboth firefly and renilla luciferase using the dual luciferaseassay system (Promega) according to manufacturer’sinstructions. All transfection experiments were conductedin triplicate and repeated three times independently.

Preparation and cytoplasm or pronuclear microinjectionof TALEN mRNA

The mRNAs were generated with T7 RNA polymerasefrom 1 lg of linearized pCS2-peas-T construct using themMessage mMachine T7 Kit (Life Technologies, 2013,Guangzhou, China) and purified using LiCl according tothe manufacturer’s instructions. Purified mRNAs werediluted to 50 ng/ll for cytoplasm microinjection) or100 ng/ll forpronuclearmicroinjection in1 mMTris.HClpH 7.5/0.1 mM EDTA and were microinjected into thecytoplasm of fertilized oocytes. A pair of primers includ-ing Ftln-F and Ftln-R were designed and used to amplifythe region containing the Foxo3 target site of the injectedzygotes (Table 1). The PCR products were sequenced inBGI (Shenzhen,China), and genemutationwas analysed.

Generation of Foxo3-targeted mice and genotyped

Foxo3 knockout mice were generated by pronucleusmicroinjectionTALEN-mRNAs inCyagen (Guangzhou,China). Genomic DNA was isolated from tail tips of

Table 1. Primers used in this study

Products Amplicon length(bp) Primer name Sequences(50?30)

Frgs / Frgs-F aattcCCTGTACGTGGCCCCTGCAGAGGCCGGAGCTGCAGGCGAGCCCGGCCAAGCCCTg

Frgs-R gatccAGGGCTTGGCCGGGCTCGCCTGCAGCTCCGGCCTCTGCAGGGGCCACGTACAGGg

Fluc / Fluc-F gatcCCTGTACGTGGCCCCTGCAGAGGCCGGAGCTGCAGGCGAGCCCGGCCAAGCCCTg

Fluc-R tcgaAGGGCTTGGCCGGGCTCGCCTGCAGCTCCGGCCTCTGCAGGGGCCACGTACAGGg

Ftln 653 Ftln-F CGGACTAGGCTCCAACAC

Ftln-R ACCTGTCCTATGCCGACC

The annealed oligonucleotides Frgs and Fluc were ligated into the pRGS and pSSA-LUC, respectively. Primers Ftln was used to amplify the region containing the

Foxo3 target site.

Foxo3 Targeted Mice with TALENs 475


Huang

高亮

founder mice. The primers Ftln-F and Ftln-R were usedto amplify the region containing the Foxo3 target sitefrom wild-type individuals or founders. The PCR prod-ucts that includedTALEN-target siteswere purified usingthe Gel Extraction Kit (Tiangen, Beijing, China) andcloned into the pEASY-T-Blunt vector using the pEASY-T-Blunt PCR Cloning Kit (Transgen, Beijing, China).Cloned plasmids were sequenced in BGI (Shenzhen,China), and gene mutation was analysed.

Histology

Ovaries were fixed for 3–5 h in 2.5% glutaraldehyde and2.5% paraformaldehyde in 0.083 M sodium cacodylatebuffer, pH 7.2, 4°C. They were then washed in 0.1 M

sodium cacodylate buffer, pH 7.2, for 24 h beforeembedding in JB-4 (glycol methacrylate) plastic (Poly-sciences, Inc., Warrington, PA, USA). Sections, 2 lmthick, were stained with haematoxylin and eosin.

(a)

(b)

(c)

Fig. 1. FOXO3 genomic structure,TALEN binding sites and reportplasmids structure. (a) Genomicstructure of the mouse FOXO3(upper panel) with an enlargementof exon 2 (lower panel), showing thebinding sites of for TALEN togetherwith the binding sites of the PCRprimers, Ftln-F and Ftln-R used togenotype the mutant alleles. TheFOXO3 TALEN target sequence,with Left and Right TALENmonomer binding sites underlinedwere shown. The TALEN sites werehigh conservative homologousregion among pig, bovine, buffaloand mouse. (b) Outline of thenuclease reporter pRGS assay.TALEN-FOXO3 targetedsequences were cloned into thelocations between the report geneRFP and eGFP, Reporterplasmid co-transfection of theTALEN-FOXO3 vectors intoHEK293T cells led to nucleasedependent gene repair and eGFPwas expressed, as shown by greenfluorescence. The red fluorescencefrom RFP was used as an indicatorof plasmid transfection efficiency.(c) Outline of the nuclease reporterpSSA-LUC assay. TALEN-FOXO3targeted sequences were cloned intothe locations between the luciferasegene segments. Reporter plasmidco-transfection of theTALEN-FOXO3 vectors andpRL-TK into HEK293T cells led tonuclease dependent gene repair andluciferase was expressed. The renillafluorescence was used as anindicator of plasmid transfectionefficiency

476 P Zhu, Q Liu, S Liu, X Su, W Feng, X Lei, J Liu, K Cui, B Huang and D Shi


Sections were analysed at 100 times magnificationunder light microscopy and photographed with digitalcamera.

Statistical analysis

This Foxo3 TALENS mutagenic non-homologous end-joining repair activity was used the pSS-LUC reporter

(a)

(b)

(c)

(d)

Fig. 2. In vitro testing of TALEN activity in HEK293T cells. (a) Blank control, added none plasmids, no red or green fluorescence detectived. (b)Negative control, added only Reporters pRGS, detectived red fluorescence. (c) Left, Right TALENs and Reporters pRGS were added 1 : 1 : 2,and red or green fluorescence were all detectived. (d) This FOXO3 TALENS mutagenic non-homologous end-joining repair activity were 31.3%using the pSS-LUC reporter to measure activity in mammalian cells HEK293T, Scale bar=100 lm



to measure activity in mammalian cells HEK293T, repairactivity was analysed using SPSS version 19.0 (SPSS,Inc., Chicago, IL, USA), and a p-value of less than 0.05was considered to be significant.

Results

Selection of TALEN-target site and activity assay in vitro

For exploring the possibility of target same site ofFoxo3 gene with different species, TALEN sites weredesigned for Foxo3 which were homologous among pig,bovine, buffalo and mouse (Fig. 1a). TALEN-Foxo3construct was cloned into a mammalian expressionvector and functionally validated using pRGS reporterassay or pSSA-LUC reporter assay in HEK293T cells.Firstly, we used pRGS reporter to investigate whetherthis TALEN can cleave the target sequence incorpo-rated between the RFP and GFP sequences inHEK293T cells (Fig. 1b). In this reporter, the gfpsequence was fused to the end of rfp sequence andinactivated by frameshift mutation and only expressedwhen the target sequence was cleaved by site-specificnucleases, which caused some small, frameshifting

insertions or deletions around the target sequence bymeans of error-prone non-homologous end-joiningrepair of the double-strand breaks.In this study, Foxo3-TALENs can recognize and

cleave the target DNA sequence (Fig. 2a–c) demon-strated by GFP-expressing cells only obtained fromthe treatment of co-transfection with the in vitro-transcribed Foxo3-TALENs. Then, we used a pSS-LUC reporter to measure their activity in mammaliancells. Site-specific nucleases were fused to the lucifer-ase sequence (Fig. 1c). Luciferase is expressed onlywhen the target sequence is cleaved by site-specificnucleases. By co-transfecting TALENS, pSS-LUC andrenilla, we found that the TALENS mutagenic non-homologous end-joining repair efficiency reached31.3% (Fig. 2d).

Generation and identification of Foxo3 mutant mice

Firstly, in vitro-transcribed Foxo3-TALENs mRNAswere injected into the cytoplasm of fifty-five mousezygotes by micromanipulation, and subsequently, thoseinjected zygotes were in vitro cultured into blastocyst

(a)

(b)

Fig. 3. PCR and genotypic analysis revealed mutations in 4 mouse blastocyst injected with FOXO3 TALENs. (a) Blastocyst 52 and 30 wereheterozygous, 46 and 53 were deleted 52 bp and 88 bp. (b) No mutations in others blastocyst



stage. The following PCR and genotypic analysis resultsindicated that there were four mutants among 48analysed embryos (Fig. 3a,b).

Secondly, pronuclear microinjections of TALEN-Foxo3 mRNAs (50 ng/ll) were conducted. Then, thepups were screened for the presence of mutant alleles by

(b)

(c)

(d)

(e)

(a)

Fig. 4. Created FOXO3 knockoutmice by TALEN-mediated genetargeting and sequenceinformation of FOXO3 mutantalleles. (a) Seven maturationFounders by TALEN-mediatedgene targeting. (b) Mutations of 20pups by PCR detecting. (c)Sequences of FOXO3 mutations inseven F0 mice lines. Deletions areindicated by dashes with grayhighlight and insertions arehighlighted in gray. The DNAsequences to which the TALENmonomers were designed arehighlighted in red. (d) DNAsequencing result of seven F0 micelines. (e) To determine whether theFOXO3 mutations were heritable,we screened all the founders andfound that the two male micetransmitted target site mutations totheir F1 mice, demonstratingsuccessful germline transmission.1–10, F1 female mice of NO.5 F0male, 11–19, F1 male mice ofNO.5 F0 male, 20, F1 mouse ofNO.4 F0 male, 21, NO.4 F0 male,22, NO.5 F0 male



sequence analysis of PCR products covering the tar-geted exon. Seven of 20 mice (35%) were identified asFoxo3 heterozygous mutant (Foxo3�/+) (Fig. 4,Table 2).Thirdly, sequence alignment results showedthat the Foxo3 sequences of two male mice were deleted34 or 51 bp, respectively. Those of the other female wereadded 2 bp, or deleted 15, 47 or 100 bp, respectively.

Phenotypes observation of Foxo3 mutant mice

To determine whether the Foxo3 mutations wereheritable, two mutant female mice were caged withone mutant male for 2 weeks. And the other mutantfemale was caged with a wide-type male for 2 weeks.Unfortunately, none mutant female was pregnant.Therefore, we arranged the two mutant males (Nos. 4and 5) to cage with two wide-type female, respectively;subsequently, the two female all got pregnancy. No.5mutant male produced normal number of pups com-pared to that of wild type; however, No.4 mutant maleonly got six pups (Table 3) during a long caged period.The ovary of mutant (Foxo3+/�) female mice wascollected, and no corpus luteum was observed, whichmaybe suggest Foxo3 knockdown would disrupt ovula-tion. The histological examination results showed therewere rare primordial and early follicles were observedcompared to that of wide-type female (Fig. 5).

Discussion

With the development of new and affordable methodsfor whole-genome sequencing, scientists are poised todeliver upon the promises of the genomic revolution totransform basic science and personalized medicine.However, the use of homologous recombination hasbeen hampered by several factors, including the lowefficiency at which engineered constructs are correctlyinserted into the chromosomal target site, the need fortime-consuming and labour-insensitive selection/screen-ing strategies, and the potential for adverse mutageniceffects. While current RNAi technology has enabled toincrease the efficiency of homologous recombination, itis still limited by variation in the degree and timing ofknockdown, and off-target effects. In contrast, the

ability to directly and specifically disrupt a gene ofinterest offers the possibility to perform intricate reversegenetic experiments on any gene, in any organism. In thepast decade, zinc-finger nucleases (Beerli et al. 1998,2000) and transcription activator-like effector nucleases(Boch et al. 2009; Moscou and Bogdanove 2009) hadbeen developed and applicated. They had been success-fully applied to disrupt gene function in differentorganism with high editing rates. A major drawbackof ZFNs, however, is the elaborate and time-consumingexperimental selection process (Maeder et al. 2008).Although simplified methods, such as modular assemblyand CoDA, have been reported (Sander et al. 2011), thequality of ZFNs generated by such platforms is contro-versial (Ramirez et al. 2008) or not determined yet. Inzebrafish, TALENs are proved significantly more likelyto be mutagenic and induce an average of 10-fold moremutations than ZFNs, 20% and 2% for TALENs andZFNs, respectively (Chen et al. 2013). Up to date, in thefamily of genomic editing toolbox, TALEN has shownto be an efficient, rapid, specific and economic methodwith a wide range of applications.TALEN-induced indels are often variable in length,

often leading to a frameshift. It is worth noting thatdifferent pairs of TALENs yield indels with differentefficiencies, ranging from 0% to approximately 65% inflies and 0% to approximately 50% in zebrafish (Weiet al. 2013). In other laboratories, seven TALENstargets in various endogenous zebrafish genes inducedtargeted indel mutations with high efficiencies rangingfrom 2% to 76% (Cade et al. 2012). DF Carlson foundthat TALENs could easily be manufactured and thatover half (23/36, 64%) demonstrate high activity in

Table 2. The presence of mutant alleles of 7 mice

Mouse Gender Mutations Change protein Phenotypes

4 Male Delete 47 bp Out of frame Subfertile

5 Male Delete 51 bp Delete 17 aa Normal reproduction

6 Female Added 2 bp Out of frame Infertile

8 Female Delete 15 bp Delete 5 aa Infertile

12 Female Delete 34 bp Out of frame Infertile

17 Female Delete 100 bp Out of frame Infertile

18 Female Not detected by plasmids sequencing but was mixed

picks by PCR products sequencing

Infertile

The presence of mutant alleles by sequence analysis of PCR products covering the targeted exon, and seven of these mice (35%) were identified as founders harboring

mutant Foxo3 alleles.

Table 3. The reproductive ability of NO.4 male mouse

Female and age Age of NO.4 male Caged time Born

1 (1.5 month) 1.5 month Caged for 1.5 month 3

2 (1.5 month) 1.5 month 3

3 (1.5 month) 1.5 month 0

4 (1.5 month) 3 month Caged for 2 month 0

5 (1.5 month) 3 month 0

6 (1.5 month) 3 month 0

No.4 mutant male got six pups which suggested it maybe subfertile.



primary cells. Cytoplasmic injections of TALEN-mRNAs into livestock zygotes were capable of inducinggene KO in up to 75% of embryos analysed (Carlsonet al. 2012). In our experiment, the success rate weobserved is 35% (>20%), similar to those described inmany other organisms (Liu et al. 2012; Ma et al. 2012;Panda et al. 2013). And the TALEN-based editing wasassociated with deletions in size 47, 51 and 100 bp,respectively, which are longer than others report ofsmall detections ranging in size from 1 to 7 bp (Liuet al. 2012; Ma et al. 2012; Watanabe et al. 2012). Inaddition, large deletions and inversions of sequencesthat are longer than 6 kb have been obtained in pigs bytargeting two TALEN pairs to the same chromosome(Carlson et al. 2012).In our study, mutations in blastocysts generated by

cytoplasm microinjections (8.3%) were lower than thatof generated by pronucleus microinjection (35%). How-ever, Liu et al. (2014) demonstrate that cytoplasmmicroinjection may result in a higher efficiency (85.7%)than that of pronucleus microinjection (30.4%). In 1985,Brinster (Brinster et al. 1985) observed that the integra-tion frequency of linearized DNA injected in the cyto-plasm or male pronucleus of mouse zygotes was 3.4% or25–30%, respectively. Geurts (Geurts et al. 2009)revealed that rate of successful mutagenesis of lgM geneby pronucleus or cytoplasm microinjection ZFNmRNAs into the zygotes was 13–16% or 2–13% in rat,respectively. The reasons for different abilities of DNAormRNA function after cytoplasm or pronucleus injectionmaybe caused by strain, gene or proficiency in injection.Foxo3�/� female mice have a very singular phenotype

which are initially fertile and subsequently infertile after15 weeks of age (Castrillon et al. 2003; Hosaka et al.2004). Foxo3 knockdown induced primordial oocyteactivation in pigs (Moniruzzaman et al. 2010). In ourstudy, all Foxo3 gene knockout females were infertile,

and their primordial oocytes were rare. The two mutantmales, one of them in which the Foxo3 sequence wasdeleted 51 bp was normal fertile; however, another wassubfertile in which Foxo3 sequence was deleted 34 bp. Itmaybe indicate that Foxo3 influence male reproduction,but more researches should be conducted to determinethis point..In conclusion, our study establishes that the TALEN-

mediated targeting is an efficient method for creatingheritable mutations in Foxo3 exon2 locus of the mousegenome, and also, this TALEN sites are homologous tobovine and sus, the work using these TALENs to editbovine or sus genome is being in progress (data notshown). Taken together, these data suggest thatTALEN-mediated in vivo mutagenesis might expeditethe creation of genetically engineered mouse models orlivestock and thereby help to accelerate functionalgenomic research.

Acknowledgements

This work was funded by the China Transgenic Project(2011ZX08007-003) and the National Natural Science Fund (GrantNo. 31160457 and 31401267), the Scientific Research Foundationof Guangxi University (Grant No. XGZ130880), Guangxi Natu-ral Science Foundation (Grant No. 2014GXNSFCB118003), andGuangxi Experiment Centre of Science and Technology(YXKT2014005).

Conflict of interest

None of the authors have any conflict of interest to declare.

Author contributions

Peng Zhu and Qingyou Liu designed and conducted the experiment,Shuai Liu, Xiaoping Su, Wanyou Feng, Xiaocan Lei, Kuiqing Cui andJinfeng Liu conducted part of experiments, Ben Huang and DeshunShi analysed data and drafted manuscript.

(a) (b)

Fig. 5. Histological analysis ofFoxo3a–/+ and control ovaries at20 weeks. (a) Histology of wide-type mouse ovary. (b) Histology ofnumber 12 female founder ovary.Green arrows: small follicles

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Submitted: 10 Nov 2014; Accepted: 23 Feb

2015

Author’s address (for correspondence):

B Huang and D Shi, State Key Laboratoryfor Conservation and Utilization of Sub-tropical Agro-Bioresources, Guangxi Uni-versity, Nanning, Guangxi 530005, China.E-mails: [email protected] (BH);[email protected] (DS)



Generation of Foxo3‐targeted Mice by Injection of mRNAs ...Ÿº因敲除小鼠... · Generation of Foxo3-targeted Mice by Injection of mRNAs Encoding Transcription Activator-like

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