Proc. Natl. Acad. Sci. USA

Vol. 89, pp. 6943-6947, August 1992 Genetics

Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice (whey acidic protein gene/A-elements/chromatin/genetic domains/mammary gland)

ROBERT A. MCKNIGHT*, Avi SHAMAY*, LAKSHMANAN SANKARAN*, ROBERT J. WALLt, AND LOTHAR HENNIGHAUSEN* *Laboratory of Biochemistry and Metabolism, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20982; and tU.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20725

Communicated by Richard D. Palmiter, April 28, 1992 (received for review February 26, 1992)

strain (16). The HindIII linker in the Kpn I site in the 5'-untranslated region enabled us to distinguish between endogenous and transgenic WAP RNA (9) and to distinguish the endogenous from the transgenic WAP genes. The chicken lysozyme 5' MARs were isolated as a 3-kb BamHI-Xba I fragment (8). Seven hundred and seventy-two zygotes were injected with the WAP transgene and transferred into 36 foster mothers, of whom 18 gave birth. The transgene was detected in 19 (17%) of the 111 pups born. Nine hundred and eighteen zygotes, coinjected with the MAR and WAP gene fragments (in a 2:1 molar ratio), were transferred into 41 foster mothers. Twenty-two of those recipients gave birth to 152 pups. The WAP transgene was detected in 27 (15%) of the pups born. Transgenic mice were identified by using PCR analysis. The first exon of the endogenous and transgenic WAP genes was amplified (9) and restricted with either HindIII or Kpn I. Transgenic mice, identified by successful HindIII digestion of the PCR fragment, were further analyzed by Southern blot analysis. The WAP 5'-probe was a 1.1-kb Sst I fragment spanning promoter sequences from -1450 to -350. The MAR 5'-probe was a subcloned 0.5-kb BamHI-HindIII fiagment, and the 3'-probe was a subcloned 1.3-kb HindIII-Xba I fragment (7, 8). Transgene copy number was determined by using PCR. The first exon of the endogenous and transgenic WAP genes was amplified from 5-fold serial dilutions of template DNA for 25 cycles in 50-,ul reaction volumes. A single 32P-end labeled primer at 3.6 x 10-2 Amol was added with 3.6 Imol ofeach unlabeled primer. Equivalent samples of each dilution were digested with Kpn I and HindIII, which restricts the endogenous and transgenic alleles, respectively. The digests were electrophoresed in 2% agarose gels, and counted with an Ambis 8-counter. Analysis of Transgene Expression. Expression of WAP transgenes was initially examined within 48 hr after parturition. Transgene expression during pregnancy, lactation, and involution was analyzed in five WAP/MAR lines. WAP transgene expression during pregnancy and lactation in mice carrying only WAP transgenes was established recently in six lines (9, 13), and involution was examined in two of these lines. Total RNA was isolated from mammary tissue (17) and 20 pg of total RNA was separated in formaldehyde gels, blotted onto a nylon membrane, and hybridized with 32plabeled oligonucleotides complementary to the transgene or endogenous WAP RNA or f3-casein mRNA (9). Mammary organ explants were prepared as described (10). Concentrations of hormones in organ culture were 100 ng/ml for insulin and hydrocortisone and 1 tkg/ml for prolactin.

Matrix-attachment regions (MARs) may ABSTRACT function as domain boundaries and partition chromosomes into independently regulated units. We have tested whether MAR sequences from the chicken lysozyme locus, the so-called A-elements, can confer position-independent regulation to a whey acidic protein (WAP) transgene in mammary tissue of mice. In the absence of MARs, expression of WAP transgenes was observed in 50% of the lines, and regulation during pregnancy, during lactation, and upon hormonal induction did not mimic that of the endogenous WAP gene and varied with the integration site. In contrast, all 11 lines in which WAP transgenes were juxtaposed to MAR elements showed expression. Accurate position-independent hormonal and developmental regulation was seen in four out ofthe five lines analyzed. These results indicate that MARs can establish independent genetic domains in transgenic mice.

Expression of transgenes is, in general, unpredictable and varies with the chromosomal site of integration. This position effect may reflect the organization of chromatin into topologically constrained loops that serve to define functional genetic domains (see refs. 1-3). Such loops are thought to be established by matrix- or scaffold-attachment regions (MARs or SARs). MARs have been identified at the boundaries of functional transcription units from several species (1, 4-6), and they have been shown to buffer effects of flanking chromatin in stably transfected cell lines (7, 8). However, whether MARs can buffer eukaryotic genes in transgenic animals is unknown. The whey acidic protein (WAP) gene is a good candidate for testing the possible function of MARs because its developmental and hormonal regulation in transgenic animals is highly position dependent. The WAP gene encodes a major milk protein, and its expression during pregnancy, lactation, and involution is regulated by multiple developmental and hormonal signals (9, 10). Expression of WAP transgenes has resulted in mammary-specific transcription, but levels were variable, and the transgenes were activated precociously during pregnancy (9, 11-14) and, in some cases, turned off during lactation. In addition, transgene expression was constitutive and hormone-independent (9, 10). Here, we report on the ability of a chicken lysozyme MAR to buffer a mouse WAP transgene from position effects in transgenic mice.

EXPERIMENTAL PROCEDURES Generation of Transgenes and Mice. The 7-kb mouse WAP transgene was identical to that used earlier (15), except that it contained the WAP protein-coding region from the YBR The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: MAR, matrix-attachment region; WAP, whey acidic protein. 6943

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Proc. Natl. Acad. Sci. USA 89 (1992)

McKnight et al.

Table 1. Expression and regulation of WAP transgene compared with endogenous WAP Regulation WAP Expression, % transgene Line copy no. Total* Per copyt Developmental* Hormonal§ Group I 8 Yes NT 7442 3 25 Yes 1 Yes 7449 50 56 NT 2 NT 9 21 7499 NT NT 27 9 7603 3 NT NT 5

Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice.

Matrix-attachment regions (MARs) may function as domain boundaries and partition chromosomes into independently regulated units. We have tested whethe...
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