Genetic Analysis: Biomolecular Engineering

15 (1999) 175–178

The use of yeast artificial chromosomes in transgenic animals:expression studies of the tyrosinase gene in transgenic mice

Patricia Giraldo, Estela Gimenez, Lluıs Montoliu *Departamento de Biologıa Molecular y Celular, Centro Nacional de Biotecnologıa, Campus de Cantoblanco, 28049 Madrid, Spain

Abstract

Variegation and inherited somatic mosaicism has been observed in transgenic mice carrying yeast artificial chromosomes(YACs) in which a DNAse I hypersensitive site (HS) located −12 kb upstream of the mouse tyrosinase gene had been deleted.At present, we are generating new transgenic animals with minor deletions of the HS. © 1999 Elsevier Science B.V. All rightsreserved.

Keywords: Tyrosinase; Albinism; YACs; LCR; Variegation

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Standard transgenic constructs, which normally con-sist of a promoter region driving the expression of acDNA cassette, often fail to recreate a fully functionaland properly regulated locus in transgenic animals [1].These phenomena are currently interpreted as the resultof position effects, related to the site of integration ofthe transgene in the host genome [2]. Some studies havereported the capability of transgenic constructs to over-come these position effects giving rise to copy-numberdependent and position-independent expression of thesetransgenes. In all these cases, specific sequences werefound to behave as locus control regions [3,4] or toinsulate the transgene from unpredictable effects ofneighbouring sequences [5,6]. These regulatory se-quences can be located far from the structural body ofthe gene and, thus, they are normally absent in thestandard transgenes. Yeast artificial chromosomes(YAC) transgenic experiments carried out with a greatvariety of genes suggest that expression from transgenesis copy number-dependent and integration-site indepen-dent [7–10]. All these findings support the concept ofgenes being organised on chromosomes as independentunits, called ‘expression domains’ [11,12].

We have applied YAC transgenesis to study theregulation of tyrosinase gene expression. Tyrosinase isthe key enzyme in the biosynthesis of melanin. It is only

expressed in two cell types, the neural-crest-derivedmelanocytes and the retinal pigmented epithelium(RPE), derived from the optic cup [13]. The tyrosinasegene maps with the classical albino mutation. In mice,a single point-mutation in exon 1 results in the accumu-lation of non-functional protein and is the primarycause of albinism [14]. Several groups reported therescue of the albino phenotype in transgenic mice carry-ing different tyrosinase minigene constructs [15,16].However, those transgenic animals showed variable andgenerally weak levels of pigmentation. The expressionwas improved when YAC tyrosinase constructs wereused to generate transgenic mice [10]. These transgenicanimals were indistinguishable from wild-type mice,with position-independent and copy-number dependentexpression of the transgene [10,20]. Thereafter, a cell-specific enhancer was found within a DNaseI hypersen-sitive region (HS) 12 kb upstream of the mousetyrosinase promoter [17,18]. The in vivo relevance ofthis HS sequence could be demonstrated with a set ofYAC tyrosinase transgenes carrying deletions of the HSregion, obtained by homologous recombination in yeastcells [19]. YAC constructs in which the HS region wasdeleted gave rise, in transgenic mice, to weak expressionlevels. Thus, it was concluded that this HS regionbehaved as a locus control region (LCR) in that itcommands the functional status of the tyrosinase ex-pression domain, protecting it from position effects[19].

* Corresponding author. Tel.: +34-91-5854844; fax: +34-91-5854506.

E-mail address: montoliu@cnb.uam.es (L. Montoliu)

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P. Giraldo et al. / Genetic Analysis: Biomolecular Engineering 15 (1999) 175–178176

A closer inspection of the weakly pigmented trans-genic mice (such YRT4) revealed the appearance ofpigmented patches, thus suggesting a variegated pheno-type [19]. To verify if the inherited somatic mosaicismobserved in skin and fur extended to the other cell typesknown to express tyrosinase [namely: retinal pigmentedepithelium (RPE) in the retina] we prepared whole-mounted retinas [21] isolated from YRT4 transgenicmice.

Fig. 1 shows a microphotography of the RPE froma representative YRT4 transgenic mice (line c2138,Ref. [19]). Direct observation of the monolayer ofRPE cells shows the occurrence of pigmented(appearing dark and contrasted, as a result of accumu-lation of melanin granules) and albino RPE cells (ap-pearing as empty areas in the microscopic preparation).Comparable variegation levels were found in otherindependent YRT4 transgenic mouse lines. Thepresence of groups of pigmented and albino cells inthe RPE suggest that variegation might be the resultof stochastic and clonally inherited patterns of ex-pression. Such variegation is not detectable in YRT2or YRT3 transgenic mice (carrying the entire HS re-gion, not shown) [19]. A detailed description ofthe variegated phenotype found in the visual systemsof these transgenic mice is going to be reported else-where.

Variegation has also been observed before inmice with a number of other transgenes [24,25]. Amongthem, the human CD2 locus [26] and the a-globingenes [27] have been shown to undergo variegationat the single-cell level when the transgenic constructswere carrying deletions within the LCR regionof the CD2 gene [26], or the aHS-40 enhancer of the

a-globin gene [27]. The presence of dominantcontrol sequences suppressed the variegation of thetransgenes. Similar patterns were seen in YRT4YAC-tyrosinase trans-genic mice. Taken together,these data suggest that the presence of an enhancerregions (or and LCR) can overcome the otherwisevariegated status of a linked transgene. Theseresults are in agreement with a model of action ofenhancers/LCR, in which these elements will act byincreasing the proportion of cells expressing a linkedtransgene (thus maintaining active the expression do-main) rather than operating at the transcriptional rate[28].

The analysis of the first set of deletions created fromthe original 250 kb YAC-tyrosinase transgene (calledYRT2) revealed the presence of LCR activity within theHS region located −12 kb upstream of the gene [19].However, this analysis could not show with precisionthe role of specific sequences found within the HS inthe LCR function because the deletions and substitu-tions made involved more than 500 bp.

A previous report demonstrated the binding of atleast two protein complexes within the core 200 bpsequences of the HS region [18]. In this study we havedevised a strategy to test, independently, the in vivorole of these two core sequences (herein referred as boxA and B) by applying the pop-in/pop-out method[22,23]. The aim is to substitute a wild-type sequence bythe desired mutated version without affecting the rest ofthe gene and without retaining the selectable markerused in the homologous recombination step, thus re-sulting in a clean mutation. We have generated YACYRT2 derivatives in which either box A or B (or both)is specifically deleted without affecting the rest of the

Fig. 1. Microphotography of a whole-mounted retina from a representative YRT4 transgenic mouse (line c2138) focused at the level of retinalpigmented epithelium (RPE) monolayer of cells. Pigmentation can be directly seen as an accumulation of melanin (black granules) within the cells.Albino (non-tyrosinase expressing) RPE cells appear as empty areas in this non-stained preparations. Magnification: 400× .

P. Giraldo et al. / Genetic Analysis: Biomolecular Engineering 15 (1999) 175–178 177

Fig. 2. Generation of YRT2DB to illustrate the pop-in/pop-out method applied for the generation of new YAC-tyrosinase derivatives, with specificdeletions within the HS (LCR) region. YAC DNAs are shown as thick lines. Black triangles indicate YAC vector arm sequences. The five exons(small open boxes) and promoter (arrow) of the tyrosinase gene are identified. Two small black boxes upstream of the tyrosinase promoter indicatesequences A and B within the LCR region. Yeast selection markers used throughout the steps are shown in italics. D stands for restriction enzymeDrdI. The relative position of the specific probe used for Southern blot analysis (see Fig. 3) is shown as a hatched box and as been repeated beloweach corresponding region of homology. (1) Starting YAC, YRT2, with the wild type LCR sequences (A and B are present). (2) YAC YRT2’,first YRT2 derivative prepared by disruption of the ura3 gene from the YAC vector arm by homologous recombination in yeast cells with a newselectable marker (lys2). Below is shown the LCR region with boxes A and B along with the restriction sites used in the Southern blot analysis(see Fig. 3). (3) Scheme of the plasmid pHSDB which carries a specific deletion of box B. Homologous sequences are shown as grey boxes. Uponlinearisation, the plasmid integrates by homologous recombination (indicated with a cross) in YRT2%. (4) Pop-in event. YRT2DBi represents aYRT2% derivative obtained by homologous recombination with the plasmid pHSDB. Below is shown a linear map with the duplication of the LCRregion. (5) Pop-out event. YRT2DB corresponds to the final step of mutagenesis. The selection against ura3 favours those homologousrecombination events (indicated by a cross in 4) that loop out the marker along with the extra copy of the LCR region.

HS region. Fig. 2 explains by example the generation ofYRT2DB (in which box B has been deleted). Theproper analysis of the different steps can be tested bySouthern blot analysis (Fig. 3). These YACs are nowbeing transferred to the germ-line of genetically albinomice in order to evaluate, separately, the in vivo contri-bution of box A and B in the establishment of the LCRactivity and the tyrosinase expression domain. Thisapproach can be applied to the analysis of the expres-sion of any gene cloned within a YAC and illustrateshow specific modifications can be brought into the genewithout affecting the rest of the locus.

Acknowledgements

The authors are grateful to Gunther Schutz,Thorsten Umland, Glen Jeffery and Soledad Montal-ban for their useful help and cooperation. The workwas supported by a Spanish Grant from CICYT, PlanNacional I+D (BI097-0628), a collaborative researchproject between Spain and United Kingdom (HB1997-0082) and funds from Laboratories Dr Esteve S.A.Patricia Giraldo is a recipient of a fellowship fromPFPI (Spanish Ministry of Education and Culture).Estela Gimenez is a recipient of a fellowship fromCSIC/Laboratories Dr Esteve S.A.

P. Giraldo et al. / Genetic Analysis: Biomolecular Engineering 15 (1999) 175–178178

Fig. 3. Southern blot analysis of DNAs from an albino NMRI mouse(lane A), YRT2% (lane B), one positive pop-in clone YRT2DBi (laneC) and two positive pop-out clones YRT2DB (lanes D and E)digested with DrdI and hybridised with a LCR-specific probe(hatched box in Fig. 2). The indicated size of hybridisation signalscorrespond to the expected DrdI fragments, as shown in Fig. 2.

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