Selective transcription of rat proenkephalin fusion genes from the spermatogenic cell-specific promoter in testis of transgenic mice.

S.A Zinn,K.M Ebert,N.D Mehta,J Joshi,D.L Kilpatrick

doi:10.1016/s0021-9258(18)54361-1

Abstract

The rat and mouse proenkephalin genes each contains two distinct promoters, one of which is utilized exclusively by spermatogenic cells. The germ cell-specific promoter lacks TATA sequences, is G+C rich, and contains multiple initiation sites. To investigate the nature of the cis-acting elements that determine selective transcription of the proenkephalin gene in male germ cells, two rat proenkephalin-chloramphenicol acetyltransferase fusion genes containing the two different promoter regions as well as 1.6 or 0.3 kilobases, respectively, of 5'-flanking sequence were expressed in transgenic mice. Multiple transgenic lines were developed which expressed the fusion genes in testis, brain, and heart but not in tissues that do not normally express the proenkephalin gene. Fusion gene transcripts in transgenic mouse testes were localized to those spermatogenic cell types that utilize the spermatogenic cell promoter and were selectively and accurately initiated from the multiple rat germ cell start sites. Transgenic mice thus provide a useful model for the localization and characterization of cis-acting elements mediating transcription of the proenkephalin gene from its germ cell-specific promoter.

Highlights

The rat and mouse proenkephalin genes each con- spermatogenic cells as well as inselected somatic tissues such tains two distinct promoters, one of which is utilized as brain and heart[5,6,7,8]
Gene expression in spermatogenic cells exhibits several unique features that appear to reflect the specialized requirements of sperm development [1,2].For instance, the expression of many genes in spermatogenic cells is regulated in a transgenic mice which appropriately express rat proenkephalin-chloramphenicol acetyltransferase (CAT)fusion genes in spermatogenic cells
Generation of Transgenic Mice Containing the RPKCAT2.3 Fusion Gene-Initial transgenic studies employed a rat proenkephalin-CAT fusion construct (RPKCAT2.3; Fig. 1)that contained a relatively largesegment (1.6 kb) of proenkephalin 5'-flanking sequence to increase the likelihood of obtaining appropriatespermatogenic cell expression of the injected gene

Summary

RESULTS

Generation of Transgenic Mice Containing the RPKCAT2.3 Fusion Gene-Initial transgenic studies employed a rat proenkephalin-CAT fusion construct (RPKCAT2.3; Fig. 1)that contained a relatively largesegment (1.6 kb) of proenkephalin 5'-flanking sequence to increase the likelihood of obtaining appropriatespermatogenic cell expression of the injected gene. Ten transgenic mice were identified from a total of 78 offspring usinga CAT cDNA probe. Four of these 10 founder (Fo)animals (lines 28,58,59,61)successfully transmitted the fusion genes to subsequent generations ina Mendelian manner. Expression of RPKCAT2.3in Tissues from Transgenic Mice-The proenkephalin gene is expressed to a relatively high degree in brain, testis, and heart of the rat and mouse [5, 10, 21]. End of the CAT coding sequence was used to determine the pattern of RPKCAT2.3 expression in transgenic mouse tissues by nuclease protection (Fig. 1).Fo animals for the six transgenic lines as well as two male transgenic founders that did not transmit toffspring (lines and70) were examined. RNAprobes and mouse testis, the 1.7-kb germ cell mRNAs are developused in nuclease protection studies are shown above the construct. mentally regulated they are first detected in spermatocytes, The different gene regions are not drawnto scale

Proenkephalin Fusion Gene Expression in Transgenic Mice

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The present data suggest that RPKCAT transgenes are DISCUSSION