Targeting iCre expression to murine progesterone receptor cell-lineages using bacterial artificial chromosome transgenesis

TECHNOLOGY REPORT

Targeting iCre Expression to Murine ProgesteroneReceptor Cell-Lineages Using Bacterial ArtificialChromosome TransgenesisAtish Mukherjee,1 Selma M. Soyal,2 David A. Wheeler,3,4 Rodrigo Fernandez-Valdivia,1

Jonathan Nguyen,1 Francesco J. DeMayo,1 and John P. Lydon1*1Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas2Department of Internal Medicine, Krankenhaus Hallein, Hallein, Austria3Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas4Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas

Received 28 August 2006; Revised 2 October 2006; Accepted 15 October 2006

Summary: Gene-targeting in embryonic stem cells hasbeen the dominant genetic approach when engineeringmouse models to query the physiologic importance ofthe progesterone receptor (PR). Although these modelshave been instrumental in disclosing the in vivo signifi-cance of the progesterone signaling pathway, genera-tion of such mice exacts considerable expenditure oftime, effort, and expense. Considering the growing listof new PR mouse models that are urgently required toaddress the next questions in progestin biology, bacte-rial artificial chromosome (BAC) recombineering in con-junction with transgenesis was evaluated as an alterna-tive method to accelerate the creation of these modelsin the future. Using this approach, we describe the gen-eration of three PR-BACiCre transgenic lines in which im-proved Cre recombinase (iCre) was targeted in-frame,downstream, and under the control of the PR promotercontained within a BAC transgene. Crossing with theROSA26R revealed that the PR-BACiCre transgenic ex-presses active iCre only in cell-lineages that express thePR. The specificity of the PR-BACiCre transgene not onlyunderscores the importance of BAC-mediated transgen-esis as a quick, easy, and affordable method by which toengineer the next generation of PR mouse models, butalso provides a unique opportunity to investigate tran-scriptional control of PR expression as well as PR struc-ture-function relationships in vivo. genesis 44:601–610,(2006). Published 2006 Wiley-Liss, Inc.y

Key words: progesterone receptor; bacterial artificial chro-mosome; iCre; transgenic

INTRODUCTION

For over a decade, homologous recombination (or gene-targeting) in murine embryonic stem (ES) cells has beenthe method of choice by which to generate mouse modelsas tools to understand the physiologic importance of the

progesterone receptor (PR) (Fernandez-Valdivia et al.,2005). Using this approach, a PR knockout (PRKO) mousewas generated (Lydon et al., 1995), in which both iso-forms of PR (PR-A and PR-B) were simultaneously ablated.Subsequent use of cre-loxP engineering strategiesallowed for the creation of PR isoform specific knock-outs, in which PR-A and PR-B were selectively abrogatedto generate PR-AKO and PR-BKO models, respectively(Mulac-Jericevic et al., 2000, 2003). More recent ‘‘knock-in’’ approaches enabled the insertion of heterologousgenes (i.e., the LacZ reporter or Cre recombinase)downstream and under the tight control of the endoge-nous murine PR promoter (Ismail et al., 2002; Soyalet al., 2005). Recently, gene-targeting approaches havealso been employed to construct a mouse model thatharbors a floxed (or conditional) PR allele to facilitateselective knockout of PR function in a tissue or cell-type specific manner (Hashimoto-Partyka et al., 2006).

Collectively, the above-mentioned mouse models havefurnished unprecedented insights into the pleiotropiceffects of the PR on the hypothalamic-pituitary-ovarianaxis, mammary morphogenesis/tumorigenesis, behavior,glucose homeostasis, parity-induced thymic involution,and the vascular system (Fernandez-Valdivia et al.,

* Correspondence to: John P. Lydon, Department of Molecular and

Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX

77030.

E-mail: [email protected]

Contract grant sponsors: National Institute for Child Health and Disease

(NICHD), National Institutes for Health (NIH); Contract grant number:

HD42311; Contract grant sponsor: National Cancer Institute; Contract grant

number: CA077530; Contract grant sponsor: Susan G. Komen Breast Cancer

Program; Contract grant number: BCTR-0503763.yThis article is a US government work and, as such, is in the public do-

main in the United States of America.

Published online in

Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/dvg.20257

Published 2006 Wiley-Liss, Inc. genesis 44:601–610 (2006)

2005). Although these mouse models have been essen-tial in establishing the in vivo significance of the PR intissue homeostasis and tumorigenesis, the cost, effort, andlength of time required to generate such models representa significant drawback as a routine genetic approach.

Because bacterial artificial chromosomes (BACs) can(in most cases) accommodate intact mammalian geneswith their full repertoire of controlling elements, can bemodified with facility and precision at base-pair resolu-tion by homologous recombination in bacteria, and canbe isolated with common DNA purification procedures,transgenics based on BAC technology offer enormouspotential as an alternative avenue to conventional gene-knockout/knockin approaches (Copeland et al., 2001).With their capacity for large genomic inserts (up to 300kb), clonal stability, and lack of chimerism, BAC-medi-ated transgenesis can, in many cases, accuratelyrecapitulate the endogenous pattern of gene expressionin an integration site-independent manner.

We have isolated a murine BAC, which contains thecomplete PR transcriptional unit with associated controlelements. Using a BAC recombineering strategy (Yuet al., 2000), Cre recombinase was knocked into Exon 1of the Pgr gene to generate a modified PR BAC, in whichCre was inserted downstream of the PR promoter.Through BAC transgenesis, we demonstrate that Creactivity is tightly modulated by the PR-BAC promoter inprogestin target tissues of the PR-BAC transgenic. Theseresults not only provide proof of principle that BACrecombineering is a feasible alternative approach bywhich heterologous genes can be efficaciously targetedto the murine Pgr locus, but also suggest that thisaffordable method could easily be applied to quicklyintroduce deletions, insertions, and/or point mutationsin the Pgr locus to study Pgr gene expression controland structure/function relationships in vivo.

Of the six murine BACs that were identified to containthe Pgr gene, the BAC clone, RP23-422-I-15 (PR-BACfrom hereon), was chosen due to the central position ofthe Pgr gene within the BAC, which allows for signifi-cant stretches of 50 and 30 flanking regions to be accom-modated within this construct; importantly, these regionsdo not contain additional genes (Fig. 1a). To reduceepigenetic silencing, the improved Cre (iCre) was usedfor PR-BAC modification (Shimshek et al., 2002); theconventional Cre gene was used to generate our previ-ously reported PRCre knockin model (Soyal et al., 2005).Standard recombineering techniques in bacterial hostswere employed to insert the iCre gene into Exon 1 ofthe Pgr gene contained within the BAC to generate thePR-BACiCre construct (Fig. 1b–c; Materials and Methods).

The iCre insertion strategy was based on the gene-targeting design used to generate our previously de-scribed PRLacZ knockin mouse (Ismail et al., 2002), inwhich the LacZ reporter gene-encoding b-galactosidase(b-gal)-was ‘‘knocked-into’’ Exon 1 of the endogenousPgr gene. Importantly, this targeting strategy maintainsthe complete complement of transcriptional control ele-ments (including cis regulatory sequences for the estro-

gen receptor-a (ER)) within the PR promoter (Hagiharaet al., 1994; Kraus et al., 1994).

Using standard BAC transgenic approaches (Marshallet al., 2004), three independent transgenic lines weregenerated, which carried the PR-BACiCre transgene(Fig. 2). Southern analysis revealed multiple copies ofthe PR-BACiCre transgene in two of these transgenic lines(1163 and 1150), whereas one copy of the transgenewas carried by the transgenic line: 1153 (Fig. 2b). Toevaluate iCre activity in situ, progeny from the threeBAC transgenic lines were crossed with the ROSA26reporter (R26R) (Soriano, 1999), in which iCre-mediatedrecombination is predicted to irreversibly activate thetranscription of the LacZ reporter gene within theROSA26 locus. Although all transgenics demonstrated asimilar spatial activity profile for iCre (data not shown),iCre activity appeared moderately stronger in lines 1163and 1150 (presumably due to a higher copy number ofthe integrated BAC in these lines). Importantly, all threeBAC transgenics, which were maintained as hete-rozygotes for the BAC transgene insertion, were func-tionally normal. For the remainder of this report, ourcharacterization of the transgenic line 1150 (referred toas PR-BACiCre/R26R from hereon) is described.

It should be noted that because Cre-loxP excision isirreversible in the PR-BACiCre/R26R transgenic, descend-ents of PR positive cells, in which the iCre recombinaseis functional, will inherit and constantly express theR26R LacZ gene. To test the specificity of the BAC trans-genic model, the PR-BACiCre/R26R mammary gland, fe-male reproductive tract, and pituitary gland (archetypalprogestin target sites (Fernandez-Valdivia et al., 2005))were examined for PR-BAC promoter dependent iCreactivity.

Although previous PRKO studies highlighted the criti-cal importance of the P proliferative signal to mammarymorphogenesis and tumorigenesis (Lydon et al., 1999),the PRLacZ and PRCre knockin models spatiotemporallytraced the activity profile of the mammary PR promoterin situ (Ismail et al., 2002; Soyal et al., 2005). Both thePRLacZ and PRCre models detected robust mammary PRpromoter activity only in the epithelial compartment ofthe mammary gland, observations that concur with pre-vious immunohistochemical studies (Ismail et al., 2004).As expected, an identical b-gal activity profile was ob-served in the mammary gland of the adult (10-week-old)nulliparous PR-BACiCre/R26R bigenic (Fig. 3a,b). Closeexamination of X-gal-stained sections revealed that themajority of luminal epithelial cells scored positive forb-gal activity in the PR-BACiCre/R26R mammary gland(Fig. 3c). When compared with mammary PR expression,X-gal-stained mammary sections from the PR-BACiCre/R26R transgenic reveal an identical spatial expressionpattern, in which only luminal epithelial cells score posi-tive for b-gal activity (Fig. 3c–h). Closer scrutiny showsthat (like the regional expression pattern for mammaryPR) a subset of luminal epithelial cells score negative forPR-BAC promoter driven iCre activity (Fig. 3e,f). Theseresults demonstrate that the mammary expression pro-

602 MUKHERJEE ET AL.

genesis DOI 10.1002/dvg

FIG. 1. Recombineering of the PR-BAC to insert iCre into Exon 1 of the Pgr gene. (a) Diagram of the PR-BAC clone (RPCI-23-422I 15).Although not to scale, the eight exons (listed 1–8) of the murine Pgr gene are indicated by black boxes; the coding region encompasses�65293 bp. The 50 and 30 regions outside the Pgr locus span 65900 and 66443 bp, respectively. The PR-BAC clone was propagated in thepBACe3.6 vector; pertinent restriction sites are included. The PCR result below shows the presence of amplicons A-I, which arise from PCRprimers that span the exon/intron boundaries of Exon 1–8 of the mouse Pgr gene, respectively; the production of these amplicons supportsthe presence of all eight exons of the Pgr gene in the PR-BAC clone (M indicates a marker lane; PCR sequences and amplification condi-tions are described in the Materials and Methods section). (b) Recombineering strategy to target the iCre cassette into Exon 1 of the Pgrgene contained within the PR-BAC (Step 1). Note that the shuttle vector, containing the iCre and FRT-KanR-FRTcassette (flanked by �60 bparms of homology to the Pgr gene (black boxes)), inserts the iCre and FRT-KanR-FRT genes 354 bp downstream of the initiating ATGB

codon; the ATGA codon (with 370 bp of sequence) is replaced by this insertion (Step 2). The FRT-KanR-FRT cassette is selectively removedby flp recombinase when the PR-BACiCre-FRT-KanR-FRT is introduced into the 294-FLP deleter bacterial strain (Step 3). (c) Schematic showsthe PCR strategy to confirm that the iCre-FRT-KanR-FRT cassette was inserted into the PR BAC in DY380 cells. Note the PCR result in thelower left panel reveals that the recombineered PR-BAC clone produces the expected PCR amplicons: PCR-A, PCR-B, and PCR-C (Lanes1, 2, and 3, respectively; lane M denotes a marker lane), indicating that the iCre-FRT-KanR-FRTcassette was inserted in the correct locationin the PR-BAC. The lower right panel shows the PCR result after the PR-BACiCre-FRT-KanR-FRT is transferred to the 294-FLP deleter bacterialstrain. Lanes 1 and 3 are positive controls for PCR amplification of PCR-B and PCR-C products, respectively (positive controls consisted ofPR-BACiCre-FRT-KanR-FRT DNA in DY380 cells). Lanes 2 and 4 show the inability to produce PCR-B and PCR-C amplicons from the modifiedPR BAC (PR-BACiCre) following flp recombinase in the 294-FLP deleter host. This result demonstrates that the FRT-KanR-FRT cassette wassuccessfully removed from the modified PR-BAC in the 294-FLP deleter strain.

603iCRE EXPRESSION TO MURINE PR CELL-LINEAGES


file for iCre from the PR-BACiCre transgene is not onlyidentical to endogenous PR but also active in this tissue.

In the case of the PR-BACiCre/R26R female reproduc-tive tract, strong iCre activity was observed uniformlythroughout the uterine horn as well as in the oviduct

and preovulatory follicle (Fig. 4a,b). PR-BACiCre/R26Ruterine sections stained for b-gal activity clearly showstrong X-gal staining in both the luminal epithelial andsubepithelial stromal compartments (Fig. 4c,d), cellularcompartments known to express uterine PR (Tibbettset al., 1998).

Our group and others demonstrated that ovarian PRexpression is rapidly and transiently induced in granu-losa cells of the preovulatory follicle by human chorionicgonadotropin (hCG) in pubescent mice that were previ-ously treated with pregnant mare serum gonadotropin(PMSG) (Ismail et al., 2002; Robker et al., 2000); hCGand PMSG are functionally equivalent to pituitary-derivedluteinizing hormone (LH) and follicle stimulating hor-mone (FSH), respectively. As reported for the PRLacZ andPRCre knockins (Ismail et al., 2002; Soyal et al., 2005),iCre activity was not detected in the ovary of the PMSG-primed 21-day-old PR-BACiCre/R26R mouse (Fig. 4e);however, strong X-gal staining was evident in the oviduct(black arrow) at this time. After 8 h post-hCG injection,iCre activity becomes visible in X-gal-stained ovarianwhole-mounts (Fig. 4f, black arrows). After ovulation(14–16 h after hCG treatment in the mouse) and subse-quent luteinization, iCre activity is present in resultantcorpora lutea (Fig. 4g,h). As previously reported for thePRCre knockin (Soyal et al., 2005), this observation issignificant in that PR expression is not detected in themouse/rat corpus luteum and therefore highlights thePR-BACiCre/R26R transgenic (as with the PRCre/R26Rbigenic (Soyal et al., 2005)) as an approach to lineallytrace the developmental fate of mural granulosa cells asthey progress from a PR positive granulosa cell to a PRnegative luteal cell.

Whole-mount X-gal staining of pituitary glands fromadult PR-BACiCre/R26R females clearly reveals iCre activ-ity is regionally restricted to the anterior lobe (Fig. 4i,j);pituitaries from age-matched PR-BACiCre/R26R malesdid not exhibit X-gal staining (data not shown). ThePR-BACiCre/R26R pituitary staining pattern is identical topituitary X-gal staining profile previously reported forthe PRLacZ and PRCre knockin models (Ismail et al., 2002;Soyal et al., 2005). Importantly, tissues known to benegative for PR expression (spleen, liver, kidney, andheart) were shown not to stain for b-gal activity in

FIG. 2. Generation of PR-BACiCre transgenic mouse lines. (a)Schematic outlining the Southern strategy to screen for foundermice (and their progeny) carrying the PR-BACiCre transgene. Formice positive for the BAC insertion, genomic DNA digested withHindIII and hybridized with a 50 Southern probe (located in Exon 1(immediately upstream of the iCre insertion)) is expected to yield a6.0 kb band attributable to the endogenous Pgr gene as well as a3.3 kb band arising from the PR-BACiCre transgene. The shorterhybridizing band from the integrated BAC is due to a novel Hindlllsite located in the iCre cassette. Wild type (WT) siblings should onlydisplay the 6.0 kb hybridizing band. (b) A Southern shows theexpected genotypic results for WT and transgenic progeny fromthree separate PR-BACiCre transgenic founders: 1163; 1150; and1153. When compared with the intensity of the WT (6.0 kb) hybridiz-ing band in all three samples, transgenic lines 1163 and 1150 con-tain at least three and two copies of the PR-BACiCre transgene,respectively; the 1153 line contains one copy of the BAC transgene.

FIG. 3. Confinement of iCre activity to the luminal epithelial compartment of the PR-BACiCre/R26R mammary gland. (a) X-gal-stainedwhole-mount of an inguinal mammary gland from a 10-week-old nulliparous PR-BACiCre/R26R mouse reveals b-gal activity throughout thearborized ductal epithelial network; LN indicates the position of the lymph node. A higher magnification of a region denoted by the blackarrow is shown in (b). Note: b-gal activity is regionally confined to the epithelial ductal system as indicated by the black arrowhead; b-galactivity is not detected in the surrounding stromal compartment. (c) An X-gal-stained transverse section of a medial duct clearly reveals b-gal activity is restricted to the luminal epithelial compartment (blue arrow); b-gal activity was not detected in the stroma (red arrow) or in theperiductal myoepithelial compartment (black arrow). (d) A serial section of the same tissue shown in (c) stained for PR immunoreactivitydiscloses an identical spatial expression pattern as observed for iCre (compare (d) with (c)); PR is only detected in the luminal epithelial com-partment (brown arrow) and not in the myoepithelial or stromal compartments (black and red arrows, respectively). (e) and (f) are highermagnification images of longitudinal sections of similar ductal structures shown in (c) and (d), respectively. Again note: b-gal activity ((e)(blue arrow)) and PR expression ((f) (brown arrow)) are localized to the luminal epithelial compartment; the myoepithelium scores negativefor b-gal activity and PR expression (black arrow). The asterisk in (e) and (f) indicates a luminal epithelial cell that is negative for b-gal activityand PR expression, respectively. (g, h) Sections of distal tips of epithelial ducts located at the periphery of the fat-pad show b-gal activityand PR expression localized to the luminal epithelial compartment (blue and brown arrows, respectively) and not to the periductal fibroblastsor stroma (black and red arrows, respectively). Scale bar in (c) applies to (d) whereas the scale bar in (g) applies to (h).



the PR-BACiCre/R26R, confirming the specificity of thisanimal model.

Collectively, these results demonstrate that iCre ex-pression in the PR-BACiCre transgenic is as tightly con-trolled by the PR-BAC promoter as previously reported

for heterologous genes regulated by the endogenous PRpromoter. The observation that the PR-BACiCre transgenicperforms faithfully supports the conclusion that most ofthe requisite transcriptional regulatory elements are con-tained within this single BAC clone. Conversely, this fea-

FIG. 3



FIG. 4. iCre is active in a subset of cell-lineages of the PR-BACiCre/R26R ovary,oviduct, uterus, and pituitary gland. (a, b)X-gal-stained whole-mounts of the ovary (O),oviduct (*), and uterus (U) are shown for theR26R and PR-BACiCre/R26R mouse, respec-tively. Although the R26R scores negative foriCre activity (a), note strong iCre activity in thepreovulatory follicle of the ovary (black arrow-head), oviduct, and uterus of the PR-BACiCre/R26R bigenic. (c, d) X-gal-stained uterine sec-tions from the R26R and PR-BACiCre/R26Rrespectively. As expected, the R26R uterus isnegative for iCre activity (c) whereas the PR-BACiCre/R26R uterus scores positive for iCreactivity in luminal epithelial (LE) and stromal(S) compartments (d). (e) Whole-mount X-gal-stained PR-BACiCre/R26R ovary (48 h afterPMSG administration). Note: iCre activity inthe oviduct (black arrowhead) but not in theovary (red arrowhead). (f) Eight hours afterhCG administration, the PMSG-treated PR-BACiCre/R26R ovary exhibits iCre activity inthe preovulatory follicle (black arrowheads);white asterisk denotes iCre activity in the ovi-duct. (g) Twenty-four hours after hCG ad-ministration, the PMSG treated PR-BACiCre/R26R ovary shows robust expression in thecorpora lutea (black arrowheads); again, whiteasterisk indicates iCre activity in the oviduct.(h) X-gal-stained section of a corpus luteum(CL) shown in (g); note most luteal cells scorepositive for iCre activity. (i, j) X-gal-stainedwhole-mounts of the pituitary gland obtainedfrom the R26R and PR-BACCre/R26R femalemouse respectively. Although the R26R pitui-tary registers negative for iCre activity (i), thePR-BACiCre/R26R pituitary gland (j) exhibitsiCre activity only in the anterior lobe (a) butnot in the neural lobe (n). Scale bars in (c) and(e) apply to (d) and to (f–g), respectively; scalebar in (i) also applies to (j).



ture is not shared by the murine ER (Esr1) locus, whichspans a number of BAC clones (Swope et al., 2002),thereby precluding ER BAC transgenesis. Although invitro studies have identified a number of cis regulatoryregions in the Pgr locus (i.e., response elements for ER,PR, AP-1, and SP-1; Hagihara et al., 1994; Kraus et al.,1994; Petz et al., 2002; Sriraman et al., 2003) that maycontrol PR expression in vivo, these sites have yet to bevalidated in a physiological context. Examination of thePR-BAC in silico clearly reveals that many more sites forthese transcription factors could also be implicated inPR transcriptional control (Fig. 5). Demonstrating thatthis BAC region is sufficient to faithfully control PR pro-moter activity in vivo provides confidence that theseand additional regulatory elements can now be function-ally evaluated for physiological significance, possibly byfirst the judicious deletion of selected conserved regionswithin the BAC (Fig. 5), followed further by point muta-tion analysis.

In conclusion, our first line of studies demonstratesthat the PRiCre BAC transgenic operates as efficiently andspecifically as our recently described PRCre knockinmodel (Soyal et al., 2005). However, while the knockinand BAC transgenic both target Cre activity to cell-line-ages that score positive for PR expression, the followingfeatures distinguish the PRiCre BAC transgenic from thePRCre knockin: (1) instead of Cre, iCre was targeted tothe Pgr locus; (2) rather than one copy, multiple copiesof iCre (driven by the PR-BAC promoter) were insertedinto the murine genome; (3) modification of the endoge-nous Pgr locus was not required to generate the PRiCre

BAC transgenic (monoallelic inactivation of the Pgr locuswas essential to generate the PRCre knockin); and im-

portantly, (4) instead of 2 years, less than 6 months wererequired to generate the first PR BACiCre transgenicfounders at a fraction of the cost and effort that wasexpended to create the PRCre knockin.

Although the PRCre knockin continues to provide es-sential insights into PR’s action in vivo (Lee et al., 2006;Mukherjee et al., 2006), the studies described hereinsupport BAC-mediated transgenesis as an importantgenetic approach to consider when designing the nextgeneration of mouse models required to address thenext questions raised by studies on progestin biology.For example, targeting genes that encode the enhancedgreen fluorescent protein ((EGFP) and spectral variantsthereof) or the reverse tetracycline transactivator (rtTA)to the Pgr locus could easily be accomplished using BACtransgenic approaches described herein. A PR-EGFP BACtransgenic would be useful not only for cell-lineage trac-ing in vivo, but also in combination with fluorescenceactivated cell sorting (FACS), this model could be usedto isolate viable PR positive cells for studies that mayrequire microarray or transplantation approaches. Onthe other hand, the PR-rtTA BAC transgenic (in conjunc-tion with the TET-ON system (Lewandoski, 2001))would allow the turning on of a target gene of choicespecifically in PR positive or PRKO cell-lineages in themouse.

We have demonstrated that all transcriptional controlelements as well as the complete coding region of thePgr gene are contained in a single BAC clone. This find-ing suggests that transgenesis using this PR BAC clone,containing a deletion, insertion, or point mutation couldbe exploited to identify and characterize critical distaland proximal enhancer elements required to control

FIG. 5. Genomic organization and comparative analysis of the murine Pgr locus. The coordinates (nucleotide numbers: 8860593-9063389)on mouse chromosome 9 (chr. 9) denote the position of the PR BAC (RPC123-422I 15). The location of the murine Pgr gene within the PRBAC (RPC123-422I 15) is diagrammed in light blue; direction of transcription is from left to right. Four custom tracks at the top show thepositions of putative full-length (not half sites) response elements for ER, PR, AP-1, and Sp-1 detected computationally in this study (verticalbars within each track). The bars are rendered in gray scale corresponding to the relative score of the site given by the detection program(methods); darker shades represent higher scores. The track labeled ‘‘Conservation’’ is a histogram on a vertical scale of 0 to 1 indicatingthe probability of phylogenetic conservation with the mouse at each base position from the nine species listed. Gaps in the conservationtrack for each species represent the location of repeat sequences as indicated by RepeatMasker. The conservation track for each speciesdisplays conservation in a gray scale (white is 0; black is 1) for the pairwise comparison with the mouse sequence.



PR expression in a specific target tissue. Similar BACtransgenic approaches could also be used for structure-function analyses of the PR protein; an area of investi-gation that has relied heavily on in vitro studies, whichin many cases suggest no clear physiological relevance.

In addition to the murine PR BAC, we recently isolateda human (h) BAC clone, which by sequencing andin silico analyses contains the entire human PR (PGR)gene including requisite control regions. The similarstructural organization of the murine and human BACs-in terms of the central positioning of the PGR gene,which ensures the inclusion of all relevant (50 and 30)regulatory sequences in the BAC transgene for correctspatiotemporal expression in vivo-suggests that similartransgenic approaches could be applied to the hPR BACto generate a number of important mouse models forstudying hPR regulation and function in vivo. Theseinclude the generation of a ‘‘humanized’’ PR mouse, inwhich hPR is expressed in the PRKO mouse as well asmouse models to address the physiological relevance ofepigenetic modifications of the PGR locus (Sasaki et al.,2001; Xiong et al., 2005) as well as the functional signi-ficance of specific single nucleotide polymorphisms inthe PGR gene that have recently been associated withendometrial and/or breast cancer predisposition in thehuman population (De Vivo et al., 2002, 2003).

In summary, the PR BACiCre transgenic provides proof-of-principle that BAC recombineering in combinationwith transgenesis can easily be applied to effectivelytarget heterologous genes to the Pgr locus with a mini-mum of expended time, effort, and expense. Moreover,the PR BAC represents an important resource forthe future study of Pgr gene expression and receptorstructure/function relationships in vivo.

MATERIALS AND METHODS

BAC Recombineering and Transgenesis

Cloned into the pBACe3.6 vector (GenBank Accession #U80929 (Frengen et al., 1999)), the murine BAC clone,RP23-422-I-15 (RPCI-23 female mouse (C57BL/6J) BACLibrary, BAC PAC Resource Center at Children’s Hospital,Oakland Research Institute, Oakland, CA), contains aninsert size of 197.6 kb of genomic DNA from the 9qA1region of chromosome 9; a region syntenic to the hu-man PGR locus located on chromosome 11q22.1–22.3(AC020735; www.ensemble.org (Soyal et al., 2002)).Sequence, Southern, and PCR analysis revealed that theRP23-422-I-15 BAC (termed PR BAC hereon) containsthe entire coding region of the murine Pgr gene, includ-ing its transcriptional control elements. The followingPCR primers were used to confirm the presence ofall eight exons in the progesterone receptor bacterialartificial chromosome (PR-BAC) clone: 50 UTR/Exon 1junction: forward: 50-CTCTGCCCCTATCACCGGC-30;reverse: 50-GGGACCTGAGTCCAAGCGTG-30 (AmpliconA (198 bp)); Exon 1/Intron 1 junction: forward: 50-CGG-

CCTCAATGGGCTCCCGC-30; reverse: 50-CAATGTTTGG-GAGAATGGTACAC-30 (Amplicon B (300 bp)); Intron1/Exon 2 junction: forward: 50-GGGAATTCATGAGTT-CAAGG-30; reverse: 50-CTTCCATTGCCCTCTTAAAG-30(Amplicon C (374 bp)); Intron 2/Exon 3 junction: for-ward: 5-GACCCTGAACTCAAAGGTGAG-30; reverse: 50-CAAGGAGGACTGCCCCTTCTC-30 (Amplicon D (511 bp));Intron 3/Exon 4 junction: forward: 50-GATGATGAGTGA-CAGCACAGTG-30; reverse: 50-GGAGAGCAACACCGTCA-AGG-30 (Amplicon E (171 bp)); Intron 4/Exon 5 junction:forward: 50-GTTAACATGGTTCATG-30; reverse: 50-CATT-TAGGATTAGATCAGG-30 (Amplicon F (197 bp)); Intron5/Exon 6 junction: forward: 50-CAACAGGAAAGAGAG-TTCC-30; reverse: 50-GTAAGGTGCCAAGTGTCTTTAC-30(Amplicon G (288 bp)); Intron 6/Exon 7 junction: for-ward: 50-GTTAGTTGCCTAGCTCAGG-30; reverse: 50-GAT-AATGGACTGAACCTGTG-30 (Amplicon H (403 bp)); andIntron 7 and 30 UTR (primers flank Exon 8): forward:50-GCAGTTCATTCAAGGGATGC-30; reverse: 50-GACACA-TGACCTGACCATC-30 (Amplicon I 487 bp)); PCR wasperformed using pfu TurboDNApolymerase1 (Stratagene,La Jolla, CA).

In accordance with the previously described red-mediated recombineering methods (Lee et al., 2001; Yuet al., 2000), the iCre recombinase cassette was insertedinto Exon 1 of the PR BAC gene. The shuttle (or target-ing vector) contained the iCre-FRT-Kanamycin resistance(KanR) gene-FRT fragment; the drug selection cassettewas flanked by two FRT sites in the same orientation.The iCre-FRT-KanR-FRT fragment was flanked by thefollowing PR homologous arms: 50 arm: 50-GATCCAA-TTCCAGACCCCCGGAGAACAGCAGACTCTTAGACAGT-GTCTTAGACTCGTTGTTA-30 and 30 arm: 50-GTCGACGA-GCACTGGAAGGCACCGGCCAGGGAGGAGGAGTCGCA-GCCAACGCGCCGTCAGCGGCCGCTAGC-30); the seque-nces of these flanking arms reside on either side of theinsertion site in Exon 1 of the mouse Pgr gene. The loca-tion of the iCre insertion site is identical to that used togenerate our previously described PRLacZ knockin re-porter mouse (Ismail et al., 2002). Following red-mediated homologous recombination in the recombino-genic bacterial strain: DY380, the KanR cassette withinthe modified PR BAC (PR-BACiCre-FRT-KanR-FRT) was re-moved in the 294-FLP deleter bacterial host (Buchholzet al., 1996) to generate the PR-BACiCre in which Exon 1of the PR BAC gene only retains the iCre insertion. ThePCR-A, -B, and -C amplicons, used to confirm the inser-tion of the iCre-FRT-KanR-FRT cassette as well as sub-sequent removal of the FRT-KanR-FRT gene, were gener-ated using the following primers: PCR-A (forward:50-GGAGGGAGCTTTCTCTGG-30 (located 248 bp down-stream from the ATGB) and reverse: 50-CAGATCTCC-TGTGCAGCATG-30 (located in the 50 region of the iCregene)); PCR-B (forward: 50-CATAGTGATGAACTACATC-AG-30 (located in the 30 region of the iCre gene) andreverse: 50-CACTGAGCGTCAGACCAAGTT-30 (located inthe 50 region of the KanR gene)); and PCR-C (forward: 50-GACTTTCCACACCCTAACTGAC-30 (located in the 30region of the KanR gene) and reverse: 50-CCTCCAGCAG-



CTGCCGGGTGCG-30 (located 973 bp downstream ofATGB)).

PR-BACiCre DNA was purified using the NucleoBondBAC Maxi kit (BD Biosciences Clontech, Mountain View,CA) and linearized with the homing endonucleasePI-SceI (New England Biolabs, Ipswich, MA) prior to zy-gote microinjection. To generate PR-BACiCre transgenicmouse lines, the linearized PR-BACiCre construct (1 ng/ml)was microinjected into the male pronucleus of FVB/Ninbred embryos, which were subsequently implantedinto pseudopregnant (C57BL6) foster mothers usingstandard approaches (Marshall et al., 2004). Transgenicfounder mice and their progeny were identified bySouthern blotting and PCR analysis with PR and Cre spe-cific probes and primers, respectively. Transgenics, in amixed strain background of FVB/N and C57BL6, weremaintained as heterozygotes for the BAC insertion. Abasic intercross with the R26R mouse generated thePR-BACiCre/R26R bigenic. The iCre gene contains itsown initiating ATG and nuclear localization signal (NLS)(Shimshek et al., 2002); however, the LacZ reporterwithin the R26R locus does not contain a NLS (Soriano,1999).

Staining for b-Gal Activity and PR Expression

Detection of b-gal activity and PR expression by X-galstaining and immunohistochemistry, respectively, isdescribed elsewhere (Ismail et al., 2002). Using an Axio-Cam MRc5 camera, digital images were obtained ofX-gal-stained whole-mounts and sections thereof usingAxioplan 2 and Stemi 2000-C microscopes (Carl Zeiss,Jena, Germany), respectively. Captured digital imageswere initially processed using Metavue Software 4.6r9(Universal Imaging, Downington, PA); final image mon-tages were assembled using Photoshop1 CS (AdobeSystems, San Jose, CA).

Mice and Hormone Treatments

Animals were maintained in a temperature controlled(228C 6 28C) room, with a 12-h light, 12-h dark photo-cycle, and provided rodent chow meal (Purina Mills.,St. Louis, MO) with fresh water, ad libitum. A superovu-lation hormonal regimen was followed according to ourprevious report (Ismail et al., 2002). Animal care andmanipulations were approved by the InstitutionalAnimal Care and Use Committee of Baylor College ofMedicine and were in accordance with practices andprocedures detailed in the Guide for Care and Use ofLaboratory Animals (NIH publication 85-23).

Computational Techniques

The estrogen response element (ERE) set was obtainedfrom transcription factor binding sites made available inthe Dragon ERE Finder version 1.0 (http//research.i2r.a-star.edu.sg/DRAGON/TFAM/) (Pan et al., 2004). Pro-gesterone response elements (PREs) were collectedfrom the literature (DeMayo and Wheeler, unpublisheddata). Activator Protein-1 (AP-1) and Sp1 binding sites

were obtained from Quality 4 level entries in the Tran-scription Factor Database: TRANSFAC1 (Matys et al.,2006). Each set of sites, ERE, PRE, AP-1, and SP-1, wereused to generate position-specific scoring matrices(PSSM) using the program Consensus (Hertz and Stormo,1999); a PSSM encodes the frequency of each base ateach position of a given DNA element. Comparing thePSSM to a target sequence of interest enables the identifi-cation of matching elements and assigns a probabilityscore to each match. The program Patser was used tosearch chromosome 9 sequences (mouse genome March2006 build) for full-length binding sites for ER, PR, AP-1,and SP-1; sites that matched the PSSM with a log proba-bility of �8 or less were saved. A profile hidden Markovmodel (Durbin et al., 1998, 1999) was constructed usingHMMer version 2.3.2 (Eddy, 2003) and used to scanchromosome 9 for matching transcription factor bindingsites; all sites with an expected value of 0.01 or lesswere saved. Binding sites for all four transcription fac-tors were displayed at the Pgr locus on chromosome 9(one track for each factor) using the custom tracksfeature provided by the University of California SantaCruz (UCSC) Genome Browser (http://www.genome.ucsc.edu).

ACKNOWLEDGMENTS

The technical assistance of Jie Li, Yan Ying, Jie Han,Jessica Li, and Jinghua Li is gratefully acknowledged. Wethank Dr. Donald L. Court, Dr. A. Francis Stewart,Dr. Rolf Sprengel, and Dr. Philip Soriano for providingthe bacterial recombinogenic strain (DY380), the 294-Flp deleter bacterial strain, the iCre recombinase, andthe R26R mouse, respectively.

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Targeting iCre expression to murine progesterone receptor cell-lineages using bacterial artificial chromosome transgenesis