Characterization of Cre recombinase models for the study of adipose tissue

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Citation Jeffery, Elise, Ryan Berry, Christopher D Church, Songtao Yu, Brett A Shook, Valerie Horsley, Evan D Rosen, and Matthew S Rodeheffer. 2014. “Characterization of Cre recombinase models for the study of adipose tissue.” Adipocyte 3 (3): 206-211. doi:10.4161/adip.29674. http://dx.doi.org/10.4161/adip.29674.

Published Version doi:10.4161/adip.29674

Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12717559

Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA P Re receptor receptor factor growth platelet-derived that reported have groups several Furthermore, vivo. in lineage adipocyte of the study further 206 Lin of WAT mass, known as lipodystrophy. as of WAT known mass, loss of obesity, or extreme characteristic WAT defining the gain, excessive from result can consequences however, metabolic severe organism; of an needs energetic the to meet dramatically shrink stasis. homeo metabolic and nutrient status, balance, of energy nance Lin including populations, precursor adipocyte white murine specific of identification Recently, the precursors. adipocyte and lations, popu cell immune various cells, endothelial cells, blood include tissue the in populations cell remaining the and tissue, adipose vivo. in tissue adipose within expression gene to manipulate developed been have that tools the on relies systems these in tissue role of the adipose to address ability our and vivo, in performed be must studies pathologies, tion. func cardiovascular and inflammation, homeostasis, glucose in Feinberg School of Medicine; Medicine; of School Feinberg http://dx.doi.org/10.4161/adip.29674 06/02/2014; 06/20/2014;Submitted: Revised: Accepted: 06/20/2014; 06/27/2014 Online: Published Email:[email protected] S Rodeheffer; to: Matthew *Correspondence s Adipose tissue is recognized as a vital player in the mainte the in player avital as recognized is tissue Adipose Mature adipocytes compose the majority of the volume of of the majority the compose adipocytes Mature

e - - :CD29 :CD29 ar 1 3-6 3 Department of of Department Section of of Section 1 ise Jeffery ise E P Re White adipose tissue (WAT) retains the ability to grow and and to grow ability the (WAT) retains tissue adipose White In order to fully understand these complex multi-organ multi-organ complex these understand order In to fully ch l s e Characterization of Cre recombinase models models recombinase Cre of Characterization is preferable for studies targeting adipocyte precursor cells vivo. in precursor adipocyte targeting studies for is preferable C do not efficiently target adipocyte precursor cells in the major adipose depots. Instead, we show that the PdgfR wethat the show Instead, depots. adipose major cells the in precursor adipocyte target efficiently not do fluorescent reporter. We find that the aP2- thatthe We find reporter. fluorescent other and these of tissue depots adipose major questioned. been recently has cytes and thataP2-and the the aP2-the α (PdgfR ar r ch e + + ap E The study of adipose tissue in vivo has been significantly advanced through the use of genetic mouse models. While While models. mouse genetic of use the through advanced significantly has been tissue vivo in adipose of study The :CD34 :CD34 R ap C e T line shows high efficiency and specificity for adipocytes, while the PdgfR the while adipocytes, for specificity and efficiency high shows T line e o r r mparative Medicine; Yale University; New New Yale University; Medicine; mparative C C re e 1,† α ll Biology; Yale University; New New Yale University; Biology; ll + + BI , Ryan Berry , Ryan :Sca-1 :Sca-1 ) is expressed on adipocyte precursor cells in in cells precursor on adipocyte expressed ) is and aP2- and Keywords: C Introduction h C icago, IL USA; USA; IL icago, + + re :CD24 :CD24 for the study of adipose tissue BI line does not efficiently target adipocytes in white adipose depots. Alternatively, Adiponectin- the depots. adipose white in adipocytes target efficiently not does line C re Cre recombinase, adipocyte, adipocyte stem cell, lineage tracing, mouse model mouse tracing, lineage cell, stem adipocyte adipocyte, recombinase, Cre 2,† Salk − + ristopher D ristopher , adipocyte progenitor cells and and progenitor cells adipocyte preadipocytes, C lines have been widely used to target adipose tissue, the specificity of these lines for adipo for lines these of specificity tissue, the adipose to target used widely have lines been 5 Yale Stem Yale Stem h 2,3 These include defects defects include These H a Boston, MA USA; USA; MA Boston, H ven, ven, C e e ll ll re we characterize were characterize H C C a e T U C † ven, ven, and Matthew SRodeheffer and Matthew nter; Yale University; New New Yale University; nter; These authors contributed equally to this work. this to equally contributed authors These 7 C r has enabled enabled has e SA; SA; h BI C urch and aP2- and T U 2 Department of Molecular, Molecular, of Department C SA; SA; 7 r rvard Medical School; Boston, MA USA MA Boston, School; Medical rvard H e lines using the membrane-Tomato/membrane-GFP (mT/mG) membrane-Tomato/membrane-GFP the using e lines dual 3 a , Songtao Yu, Songtao 4 Department ofDepartment Pediatrics; A dipocyte C Adipocyte 3:3,206–211;July/August/September 2014;©2014LandesBioscience - - - - re C Salk r colony maintenance. colony of mouse generations over several or efficacy expression Cre in for changes potential the and recombination, loxP Cre-mediated to sites genomic of different sensitivity varying the transgenes, of Cre expression erroneous the including approaches of these pitfalls and limitations the mind in to keep ever, it important is how techniques, these applying When Cre. tamoxifen-sensitive or expression Cre doxycycline-regulated as such models targeting inducible with achieved be can controlover expression gene poral with the expression of the endogenous gene. endogenous of the expression the with compared of expression Cre fidelity the affect may and transgene, Cre of the pattern expression the affect site can integration the of location the promoter and sequence of this site.length The genome at arandom into the integrated then is which fragment controlof apromoter under a “knock-in”, placed be or it can promoter, endogenous of an termed often downstream integrated be can recombinase Cre vivo. in function of tissue-specific study lineage. cellular adipocyte the tool for targeting promoter a useful is this that indicates which WAT and traces all adipocytes in normal murine WAT murine normal in depots, adipocytes all WAT traces and pose tissues. Of these, the most commonly used Cre transgenes Cre transgenes used commonly most the these, Of tissues. pose adi in of recombinase Cre expression the to drive generated been promoters have adipocyte-specific several contexts, relevant cally e recombinase activity in multiple cell populations of the the of cellpopulations multiple in activity e recombinase lines lack specificity for adipocytes within adipose tissue, adipose within adipocytes for specificity lack lines H

a The Cre/lox system of gene targeting has revolutionized the the revolutionized has of targeting gene system Cre/lox The To facilitate the study of adipocyte function in physiologi in function of adipocyte To study the facilitate ven, ven, 4 , Brett AShook , Brett C C e T U ll and Developmental Biology; Yale University; New New Yale University; Biology; Developmental and ll 2,3,5, SA; SA; C h 6 * ildren’s Memorial Research Division of of Division α - C 11,12 r 2 e sley , Valerie E E n R docrinology; Beth Israel Deaconess Medical Medical Deaconess Israel Beth docrinology; U C L a nd PdgfR nd H or C en ter; Northwestern University 2,5 α - an DRosen , C E r v e 10 E Additionally, tem Additionally, R α J - H H C a U li r V ven, ven, e line e line olume 3Issue 3 nes nes C T U - 6,7 SA; SA; C , e nter; nter; 8,9 - - - -

©2014 Landes Bioscience. Do not distribute. www.landesbioscience.com ing the adipocyte lineage in vivo. in lineage adipocyte the ing ing mature adipocytes, while the PdgfR the while adipocytes, mature ing inducible for target model auseful is Adiponectin-CreERT the PdgfR in precursors pocyte Cre labeled in both WAT both aP2-Cre in the in BATlabeled depots and were cells of endothelial percentage alarge that indicated ysis ( labeled were depot (iBAT) tissue adipose brown intrascapular the in cytes adipo of the half WAT in approximately labeled while depots, of aP2-Cre of depots adipose several in of Creexpression pattern the analyzed we When occurred. has recombination Cre-mediated which in of adipocytes identification the facilitating boundaries, cellular modelbrane-targeted, this provides fluorescently labeled clear mem are proteins fluorescent these Since (mG). eGFP geted of membrane-tar expression the permits then which cassette, tdTomato of the excision the in results sion of recombinase Cre tdTomato expres the (mT), and membrane-targeted expresses adipocyte precursors and embryonic tissues. embryonic and precursors adipocyte macrophages, cells, endothelial brain, including types, cell and tissues other in activity Cre to have shown been have lines these separate lines (aP2-Cre lines separate study of adipocyte function. of adipocyte study by Adipoq (encoded protein Adiponectin promoterthe adipocyte-specific of the adipocytes and a lack of specificity with the aP2-Cre the with of specificity alack and adipocytes and PdgfR Adiponectin-CreERT tamoxifen-inducible the as well as lines, 4 ( protein binding acid promoter the fatty from of the were created widely used in studies to target adipocytes in vivo. in adipocytes to target studies in used widely to adipose tissue. to adipose specific highly by to multiple be groups shown been have lines form lineage tracing of white adipose tissue. adipose of white tracing lineage form to per used (mT/mG) model previously we have reporter that fluorescent dual to the lines of these each we crossed lines, Cre originally shown to efficiently label adipocytes, label to efficiently shown originally was line this while that indicate data These not shown). (data mice of these cells muscle skeletal and liver of both gible labeling endothelial stain GSIB stain endothelial the with SWAT in of eGFP-positive co-stained cells majority the WAT ( depots in were not labeled populations precursor adipocyte the while ulations within adipose tissue in the aP2-Cre the in tissue adipose within ulations pose tissues. adi in activity Cre recombinase assess to quantitatively model reporter fluorescent dual amembrane-targeted using lines, Cre used of commonly specificity and efficiency recombination the to investigate we sought vivo, in lineage adipocyte the geting Fabp4 To characterize the pattern of Cre expression in the aP2- the in of Cre expression pattern the To characterize Here we characterize the recombination of specific cell pop cell of specific recombination the Here we characterize Salk lines, and remarkably low recombination efficiency in adi in efficiency low recombination remarkably and lines, ) gene which encodes adipocyte protein 2(aP2). adipocyte encodes which ) gene BI α ; mT/mG mice, we found that few adipocytes were adipocytes ; mT/mG few that we found mice, -CreERT lines. We find incomplete targeting of targeting incomplete We lines. find -CreERT Fig. Fig. 16,18,20

1A and B and 1A 1B ) has been used to generate Cre lines for the for the lines Cre to generate used been ) has ). Further confocal analysis confirmed that that confirmed analysis confocal Further ). To identify the most useful tools for tar tools useful To most the identify BI

4 ( and aP2-Cre and Fig. α Results ). Additionally, flow cytometry anal cytometry flow Additionally, ). -CreER lines. Finally, we show that we show Finally, that lines. -CreER 20,21

1C TheseAdiponectin-Cre mouse ). Finally, we observed negli we observed Finally, ). Salk α ) were generated -Cre is useful for study useful is -Cre 16-19 BI 9,22 and aP2-Cre and 14 This reporter reporter This it now targets it targets now Alternatively, 16 BI However, However, and aP2- and 14,15 BI 13 line, line, Two Two and and Adipocyte Salk ------

tissue. adipose within cells endothelial and adipocytes brown primarily a similar degree ( degree a similar ( percent 80 labeling of CD31labeling significant and cells lineage blood in some labeling observed we also depots, all in populations precursor adipocyte the in ing ( lineage in the aP2-Cre the in lineage much higher than the aP2-Cre the than much higher WAT in line was by depots this labeled of adipocytes centage mT/mG per We the reporter. the using that found fluorescent mature adipocytes, has been used by to multiple groups used been has adipocytes, mature not shown). (data mice of these muscle skeletal and liver of the cells in recombination Cre-mediated Fig. tissue depots (n=3). ( depots tissue adipose indicated the of populations cell in eGFP of expression mediated mT/mG mice stained with isolectin GSIB isolectin mT/mG with stained mice Figure 1. cytes. ( cytes. bars in ( in bars from aP2-from adipose tissue; SVF, stromal vascular fraction. vascular SVF, tissue; stromal adipose WAT; RWAT, WAT; retroperitoneal MWAT, WAT; mesenteric BAT, brown

We next characterized Cre expression in the aP2-Cre the in expression Cre We characterized next The promoter for adiponectin, a hormone secreted by ahormone secreted promoterThe for adiponectin,

2B A) A and C A and ), again indicating a lack of specificity for the adipocyte adipocyte for the of specificity alack indicating again ), aP2- C C re o nfocal microscopy of the indicated adipose tissue depots depots tissue adipose indicated the of microscopy nfocal Fig. BI ; mT/mG ( mice. C r ) are 100) are μ e

BI + 2A and B and 2A primarily labels endothelial cells endothelial and brownadipo labels primarily endothelial cells in all adipose depots analyzed analyzed depots adipose all in cells endothelial Fig. C

) 2A and B and 2A Salk C m. SWAT,m. WAT; subcutaneous GWAT, gonadal o line. However, we observed negligible negligible However, line. we observed nfocal microscopy of SWAT of aP2- microscopy from nfocal B ). Brown adipocytes were labeled to were labeled adipocytes Brown ). ) Quantification of cells displaying displaying cells of ) Quantification BI line, and varied between 50 and 50 and between varied and line, ). While there was some label was there While ). 4 to label endothelial cells. Scale Scale cells. endothelial tolabel Salk C C line line re re- 207 BI - ; - -

©2014 Landes Bioscience. Do not distribute. 208 cific to adipocytes, cific create the Adiponectin-Cre the create to BAC used same the with generated line Adiponectin-CreER a new in inducible Cre expression To vivo. in characterize cytes generate adipocyte-specific Cre lines. Cre adipocyte-specific generate Adiponectin-Cre this model, including the adipocyte precursor populations, populations, precursor adipocyte the including model, this in recombination Cre-mediated displayed SVF adipose the ( adipocytes brown in observed was 85% recombination than greater and lation, popu cell other any in recombination negligible with analyzed, WAT all in depots 100% nearly was recombination adipocyte ( observed was ing label no adipocyte treatment, tamoxifen Before forfen 6d. tamoxi 50 mg/kg with mice mT/mG treated and line reporter iue 2. Figure WAT; fraction. vascular SVF, BAT, tissue; stromal adipose brown WAT; GWAT, WAT; gonadal RWAT, WAT; retroperitoneal MWAT, mesenteric ( in bars (n=3). Scale depots tissue adipose indicated the of populations cell in eGFP of expression mediated ( tissue. from aP2-from

A) C aP2- C re o Salk nfocal microscopy of the indicated adipose tissue depots depots tissue adipose indicated the of microscopy nfocal C ; mT/mG ( mice. r e R Salk line Fig. 9,16,18 labels multiple cell populations within adipose adipose within populations cell multiple labels Fig. 12

and useful for targeting mature adipo mature for targeting useful and

3A and B 3A and has been shown several times to be spe to be times several shown been has S1A B R ), and after tamoxifen treatment, treatment, tamoxifen after and ), mice, we crossed this line to the to the line this we crossed mice, ) Quantification of cells displaying displaying cells of ) Quantification ) are 100A) are μ ). Importantly, no cells within within no cells Importantly, ). 20,21 The most widely used used widely most The m. SWAT,m. subcutaneous C A re- dipocyte - - - - - and liver are minimally targeted by the PdgfR by the targeted minimally are liver and adipocyte precursor populations within WAT, within populations precursor adipocyte mice are adipocyte precursors. adipocyte are mice WAT of PdgfR within Cre-recombination SVF displaying cells of the majority vast the addition, In mG mice. PdgfR in labeled are precursors adipocyte of the all tion in both of these tissues ( tissues of these both in tion PdgfR of liver and muscle of the cells the in recombination mediated Cre- we assessed tissues, but not major metabolic other lineage, adipocyte the targeting studies for metabolic useful be may line mature adipocytes. ( Fig. vascular fraction. WAT; MWAT, WAT; mesenteric SVF, BAT, tissue; stromal adipose brown SWAT, WAT; subcutaneous GWAT, WAT; gonadal RWAT, retroperitoneal ( in bars (n = 3). Scale depots tissue adipose indicated the displaying cells C Adiponectin- from depots tissue adipose indicated the of microscopy iue 3. Figure

re We and other groups have shown that PdgfR that We shown have groups other and E

R T; mT/mG mice after tamoxifen treatment. ( T; treatment. mT/mG tamoxifen after mice 3B α -Cre; mT/mG We-Cre; of mice. recombina low found levels ), confirming the specificity of this promoter for of this specificity the confirming ), Adiponectin- C re-mediated expression of eGFP in cell populations of of populations cell in eGFP of expression re-mediated C r e E R T labels mature adipocytes. ( matureT adipocytes. labels Fig. 9 To determine whether this Cre Cre To this whether determine

4A ), indicating that the muscle muscle the that indicating ), B promoter. These These α promoter. 8,9 ) Quantification of of ) Quantification is expressed on expressed α is and that almost almost that and α -Cre; mT/mG-Cre; V ) are 100A) are olume 3Issue 3 α A) -Cre; mT/ -Cre; C o nfocal nfocal μ m. m. -

©2014 Landes Bioscience. Do not distribute. adipocyte lineage cells in WAT in cells lineage adipocyte region known to contain PdgfR to contain known region ( skin of layer the dermal the in of GFP-positive cells and the Bergles group Bergles the and www.landesbioscience.com group Richardson the by both generated vivo. in lineage cellular adipocyte of the studies functional PdgfR the that suggest data and PdgfR the brain. the in precursors oligodendrocyte to label shown been previously have skin, in WAT PdgfR in we treated vivo, in depots precursors of adipocyte study for the lines of these of both utility treated PdgfR treated tamoxifen- In WAT mice. of these depots primary and skin the in recombination Cre-mediated displaying cells precursor pocyte of adi percentage the analyzed subsequently for 6dand daily mG mice (n=3). mice mG ( from PdgfR from the indicated depots of PdgfR of depots indicated the ( 2animals. from image Representative compartment. dermal the within layer adipocyte the of location the indicates adipocytes of staining Perilipin dermis. upper the of cells fibroblastic in bars in ( in bars Figure 4. Inducible PdgfR of versions the 25 although with variable efficiency. To assess the potential potential To the efficiency. assess variable with although A and PdgfR 23,24 α α -CreER - C The PdgfR The C α re; mT/mGre; ( mice. ) are 100) are μ α -CreER C - C ) Immunofluorescence image of the back skin of PdgfR of skin back the of image ) Immunofluorescence re E JHU R d 24 JHU ; mT/mG mice with 50 mg/kg tamoxifen tamoxifen ; mT/mG 50 mg/kg with mice (PdgfR

oes not efficiently label adipocyte precursor cells. ( cells. precursor adipocyte label efficiently not oes m. SWAT,m. WAT; subcutaneous GWAT, WAT; gonadal fraction. vascular SVF, stromal α ; mT/mG mice, we observed a high level level ; mT/mG ahigh we observed mice, -CreER α - C α B r -Cre line may be appropriate be for may line -Cre ) Quantification of cells displaying displaying cells of ) Quantification e α E α 8 and fibroblast-like cells in the the in cells fibroblast-like and -CreER R -Cre-expressing cells including including cells -Cre-expressing U UCL C D α L ; mT/mG PdgfR and ) Quantification of cells displaying displaying cells of ) Quantification -Cre mouse line have been been have line mouse -Cre line has been used to label to label used been has line JHU 23 α (PdgfR ). Both of these lines lines of these Both ). -CreER α UCL α -CreER Fig. - C ; mT/mG r e

E 4B R J H UCL Adipocyte C U ), a ), ; mT/mG mice after daily treatment of 50mg/kg tamoxifen for 6 d ( for tamoxifen 50mg/kg of ; mT/mG treatment daily after mice re-mediated expression of eGFP in liver and skeletal muscle of PdgfR of muscle skeletal and liver in eGFP of expression re-mediated ) - α C - C re-mediated expression of eGFP in the adipocyte precursor population of of population precursor adipocyte the in eGFP of expression re-mediated an adipogenic population; adipogenic an in PdgfR in of WAT fraction depots vascular stromal the we analyzed When not shown). (data were labeled cells precursor adipocyte dermal mT/mG ( mice ficient for assessing gene function in adipocyte precursors in vivo. in precursors adipocyte in function gene for assessing ficient insuf is lines these in precursors adipocyte in activity Cre that essential for the study of adipose function and metabolic disease. disease. metabolic and function of adipose study for the essential ( efficiency not recombination improve the did for 5d), mg/kg (300 dose tamoxifen ahigher with mG mice SWAT the in of PdgfR percentages recombination GWAT of both better tions SWAT, and marginally only with popula precursor adipocyte in 3%) than (less of recombination r A) e

E Tools for the genetic targeting of adipose tissue in vivo are are vivo in tissue of adipose Tools targeting genetic for the C R o U C nfocal microscopy of the liver and skeletal muscle (gastrocnemius) (gastrocnemius) muscle skeletal and liver the of microscopy nfocal L ; mT/mG mouse after tamoxifen injection showing recombination recombination showing injection ; mT/mG tamoxifen after mouse α -CreER Fig. UCL

4C ; mT/mG mice, we observed very low rates low rates ; mT/mG very we observed mice, ). Treatment). of PdgfR Discussion 25,26 - 1% intra of the however, than less Fig. α

S1C -CreER = 3–4). Scale Scale n = 3–4). α ), indicating indicating ), -CreER α - JHU C r e; mT/ e; ; mT/ ; JHU 209 - - ;

©2014 Landes Bioscience. Do not distribute. tions. 210 expression from the PdgfR the from expression 60 PdgfR the to generate used of of lack due to be the may efficiency low labeling this construct, Cre Cre function. cell and dynamics lineage of cellular studies in sue tis aparticular in of interest types cell to the Cre-recombination of specificity the of determining importance the highlight data these Additionally, over time. aline within recombination ing the dermis ( dermis the PdgfR of other tion, despite labeling bination in the PdgfR the in bination recom of alow Cre-mediated level displays muscle skeletal adult For example, not is observed. pattern expression embryonic this given expected be would that recombination broad the mice, However, PdgfR adult in cartilage. and dermis, muscle, skeletal the including tissues mesodermal several rise give which embryonic day 8, PdgfR 8, day embryonic Consistent with previous reports, nent expression occurring in mesodermal tissues. mesodermal in occurring nent expression promi most the with ectoderm, and mesoderm endoderm, of the PdgfR that shown have reporter rescent endogenous PdgfR the mT/mG reporter indicates that the aP2-Cre the mT/mG that the indicates reporter cre reporters, cre LacZ cytoplasmic using studies to previous However, contrary Cre generations, over lines these in pattern expression Cre the in due tobe ashift could WAT in this Since depots. of adipocytes majority the label in WAT-residentin APs, recombination partial to achieve of tamoxifen doses high very used one has group While cells. precursor adipocyte targeting reporter. mT/mG the coupledwith when studies pulse-labeling titative quan to perform controlnecessary temporal the provides also and effects, confounding present could tissue adipose in defects developmental which in animals of adult studies in applied be can model This inducible manner. an in adipocytes target to efficiently used be can line Adiponectin-CreER the here that precursors. PdgfR for the case the to be appears This gene. endogenous the from to differ gene trans of the expression the cause site can insertion of the effects or positional activity enhancer in differences large, is expression lineage. adipocyte the in of function gene or for study studies lineage adipocyte for quantitative not useful are they that PdgfR these in efficiency low labeling the reason, of the genome. Regardless the in sites integration in PdgfR the between of the existing PdgfR existing of the

cis We and others have previously found that the Adiponectin- the that found We previously have others and The PdgfR The Even when the promoter region used to drive transgene transgene to drive promoterEven the used when region

k BI R b upstream of the PdgfR of the b upstream effectively targets mature adipocytes, mature targets effectively line and the aP2-Cre the and line -regulatory elements that are normally necessary to drive to drive necessary normally are that elements -regulatory 23,24,27 9 Another potential factor in the difference in labeling labeling in difference the in factor potential Another During murine embryonic development, staining for development, staining embryonic murine During 12 Fig. 18 these data emphasize the importance of monitor importance the emphasize data these α quantitative analysis of adipocyte labeling with with labeling of adipocyte analysis quantitative -CreER model would also be a valuable tool for avaluable be also would model -CreER

4 ). Given that both of the promoter sequences promoter of the sequences both Given that ). α and experiments utilizing a knock-in fluo a knock-in utilizing experiments α and α -Cre line, which efficiently labels adipocyte adipocyte labels efficiently which line, -Cre -CreER and PdgfR and -CreER α 8 -CreER lines efficiently label this popula this label efficiently lines -CreER we show here that in our hands neither hands our in we show here that α is expressed throughout the somites, the throughout expressed α is -Cre line. These data suggest that the the that suggest data These line. -Cre Salk promoter in certain cell popula cell α promoter certain in α gene compared with the PdgfR the with compared α gene line are not specific to adipocytes. to adipocytes. not specific are line -CreER lack a region of at least of atleast aregion lack -CreER 16,17 our data show that the aP2- the show that data our α -expressing populations in in populations -expressing α α is expressed in regions regions in expressed α is -CreER lines indicates indicates lines -CreER -Cre lines is differences differences is lines -Cre 9,16,18,20 BI 28-31 and we show and line does not does line Starting at Starting α -Cre -Cre A dipocyte α 28 ------

gene. The PdgfR The gene. of the expression embryonic to induce required are that construct PdgfR the in elements enhancer promoter and/or populations such as the Muller glial cells of the retina, of the cells glial Muller the as such populations ( terns pat- expression embryonic early these not mimic does model Cre cell types, such oligodendrocytes, as types, cell promoter other in this from expression Cre However, known the tissues. metabolic other in effects “off target” not significant have expression of Cre from the PdgfR the of from Cre expression line adipocytes. Cre mice (013148), mice Cre PdgfR Dr described. for 5d. daily oil vegetable in tamoxifen mg/kg of 300 gavage dor oral for 6–7 daily oil etable veg in tamoxifen of 50 mg/kg injections intraperitoneal either 10 were given 5and of wk age between mice where indicated, For PdgfR sacrificed. then and one week, for to recover allowed were then Mice for 6d. oil vegetable in tamoxifen of 50 mg/kg injections intraperitoneal daily were given age. of 6 wk 4 and between were males analyzed mice all lines, CreER PdgfR and Adiponectin-CreERT for the Except Laboratories. atJackson (024671) now mice available are AdiponectinCre-ERT Laboratories. Jackson from (007676)mG mice were purchased skeletal muscle. These findings indicate that PdgfR that indicate findings These muscle. skeletal and liver the as such organs major of metabolic other cells mal parenchy the in expressed it not is significantly show that data subsets of white adipocytes of white subsets and muscle either target BAT also to target promotersthe known BAT, WAT and target targeting without to specifically known models any not currently are There adipocytes. brown and white both in recombinase Cre express models these that is function PdgfR model. this from results interpreting Cre Adiponectin- the while vivo, in depots adipose in cells lineage of adipocyte targeting for the available model best the rently by incubation with alexa fluor-conjugated secondary antibodies. secondary fluor-conjugated alexa with by incubation followed at4°C overnight 1:1000) Abcam, A(goat, perilipin and 1:1000) Abcam, (chicken, GFP against antibodies mary pri with incubated and paraformaldehyde 4% with were fixed adipocytes. (IACUC). aP2-Cre Use Committee and Care Animal University’s Institutional Kahn, obtained in October 2013. October in PdgfR The obtained Kahn,

Confocal microscopy and flow cytometry were performed as as were performed cytometry flow and microscopy Confocal mice 8-wk-old male experiments, For Adiponectin-CreERT Another caveat that must be considered when using either the the either using when considered be must that caveat Another All animal studies followed guidelines issued by Yale issued guidelines followed studies animal All R 23 Dana McTigue. aP2-Cre The Dana and Adiponectin-CreER are effective for targeting mature mature for targeting effective are Adiponectin-CreER and was independently obtained from Dr Anne Perl and Dr Anne from obtained independently was Fig. or the Adiponectin promoters for the study of adipose of adipose promoters study for the Adiponectin α or the 9,36,37

4 35 ). This restricted expression may be due to a lack of due to be alack may expression restricted This ). Therefore, we conclude that PdgfR we conclude that Therefore, For immunofluorescence, 14 μ immunofluorescence, For α Materials and Methods and Materials -Cre model has been shown to label other cell cell other to label shown been has model -Cre BI mice were a generous gift from Dr Barbara Dr Barbara from gift were agenerous mice α 32-34 -CreER or target both beige and brown brown and beige both or target Salk promoter in the PdgfR α promoter the in 23 JHU should be considered when when considered be should mice (005069), PdgfR mice (018280), and mT/ (018280), and mice α -CreER experiments, experiments, -CreER α -CreER m skin sections sections m skin V α α α olume 3Issue 3 -Cre genetic genetic -Cre -Cre is cur is -Cre -Cre should should -Cre UCL 27 but our our but mouse mouse α α α ------

©2014 Landes Bioscience. Do not distribute. 13 12 10 11 9 8 7 www.landesbioscience.com 6 5 4. 3 2 osen ED, Spiegelman BM. Adipocytes as regulators regulators as Adipocytes BM. Spiegelman ED, osen 1 DK085171 of DK078061 Institutes and National to E.D.R., grants of Health Institutes National to M.S.R., DK090489 ......

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This work was funded by National Institutes of Health grant grant of Health Institutes by National funded work was This were disclosed. of interest conflicts No potential org/10.1016/B978-0-12-411619-1.00001-X G 24; 49:219- 1992; Biochem JCell expression. gene tissue-specific adipose for requirements of analysis enhancer: cell afat of Identification BM. Spiegelman 2014; 537:1-16; 2014; Enzymol Methods models. knockout and transgenic K jcb.240490303 S M ni0707-665 8; 8:665- 2007; Immunol Nat targeting. gene ditional PMID:23823474 18:9-20; 2013; Metab Cell use. prudent their for considerations and lines driver org/10.1038/ncb2696 B 91; 15:480- 2012; Metab Cell feeding. high-fat and β by recruited genitors pro adipocyte bipotential of identification vivo In L 2013; 15:302-8; in lineage cellular adipocyte dx.doi.org/10.1016/j.cmet.2013.06.011 cmet.2012.03.009 http://dx.doi.org/10.1016/j.cell.2008.09.036 PMID:18835024 135:240-9; 2008; Cell vivo. in cells progenitor adipocyte white of Identification R M org/10.1097/01.md.0000111061.69212.59 2004; 83:18-34; (Baltimore) Medicine literature. the of review and 35 of cases report lipodystrophy: partial acquired in derangements autoimmune and metabolic and tures PMID:24798950 press; 13;In 2014; Pract Clin Res Diabetes 2diabetes. type and syndrome metabolic obesity, between alink as Inflammation N. E 57:297-311; PMID:16409151 2006; Med Rev Annu complications. metabolic of management and A dx.doi.org/10.1210/jc.2010-1378 Metab 2010; 95:5419-26; PMID:20843952 Endocrinol JClin Study. Heart Jackson the factors: risk cardiometabolic on tissue adipose subcutaneous and visceral abdominal of Impact Taylor HA. JJ, Carr L dx.doi.org/10.1146/annurev.med.57.022605.114424 ATVBAHA.107.159228 49; 28:1039- 2008; Biol Vasc Thromb Arterioscler risk. cardiometabolic global to contribution syndrome: metabolic the and obesity P.Poirier Abdominal OF, Bertrand J, Rodés-Cabau E, P, Larose Mathieu D org/10.1038/nature05483 R 2006; 444:847-53; Nature homeostasis. glucose and balance energy of chmidt-Supprian M, Rajewsky K. Vagaries of con of Vagaries K. Rajewsky M, chmidt-Supprian iu J, Fox CS, Hickson DA, May WD, Hairston KG, KG, Hairston WD, May DA, Hickson CS, iu J, Fox ee YH, Petkova AP, Mottillo EP, Granneman JG. JG. EP, Granneman AP, Mottillo Petkova YH, ee sser N, Legrand-Poels S, Piette J, Scheen AJ, Paquot Paquot AJ, Scheen J, Piette S, Legrand-Poels N, sser erry R, Rodeheffer MS. Characterization of the the of Characterization MS. Rodeheffer R, erry odeheffer MS, Birsoy K, Friedman JM. JM. Friedman K, Birsoy MS, odeheffer garwal AK, Garg A. Genetic basis of lipodystrophies lipodystrophies of basis Genetic A. Garg AK, garwal ang S, Kong X, Rosen ED. Adipocyte-specific Adipocyte-specific ED. Rosen X, Kong S, ang raves RA, Tontonoz P, Platt KA, Ross SR, SR, Ross Tontonoz P, KA, Platt RA, raves esprés JP, Lemieux I, Bergeron J, Pibarot P, Pibarot J, Bergeron I, JP, Lemieux esprés PMID:17579640 agnuson MA, Osipovich AB. Pancreas-specific Cre Cre Pancreas-specific AB. Osipovich MA, agnuson isra A, Peethambaram A, Garg A. Clinical fea Clinical A. Garg A, Peethambaram A, isra PMID:22482730 PMID:18356555 PMID:1644859 Disclosure of Potential Conflicts of Interest Conflicts ofPotential Disclosure References PMID:24480338 PMID:23434825 PMID:14747765 PMID:17167472 ; ; ; http://dx.doi.org/10.1161/ ; http://dx.doi.org/10.1016/j. 3-adrenoceptor activation activation 3-adrenoceptor http://dx.doi.org/10.1002/ http://dx.doi.org/10.1038/ Acknowledgments

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al. Lessons on conditional gene target gene conditional on Lessons al. Adipocyte ; PMID:16952017 PMID:11217863 PMID:14660788 ; ; ; http://dx.doi.org/10.1016/j. http://dx.doi.org/10.1016/j. www.landesbioscience.com/journals/adipocyte/article/29674/ here: found be may materials Supplemental from the Yale Stem Cell Center to R.B. Center Yale Stem Cell the from Fellowship Graduate Lo the and to M.S.R., JF-12-046 grant Association to V.H., Diabetes AR060295 grant American Health http://dx.doi.org/10.2337/ http://dx.doi.org/10.1210/

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