WAP based mammary expression vectors

Regulatory elements from the mouse WAP gene (Genbank U38816) have frequently been used to target the expression of transgenes specifically to mammary tissue of transgenic mice, sheep and pigs. Based on our experience and results from other labs, cDNA clones can be expressed well in WAP gene based expression vectors. We have integrated all information available and built several expression vectors which are expressed well in mammary epithelial cells during late pregnancy and lactation.

If a target gene exists as a cDNA it may be best to replace sequences between exon 1 and 3 of the WAP gene with the cDNA (Asp718 cuts in the first exon of the WAP gene immediately 5' of the ATG and SalI cuts in the 3rd exon). For this purpose three new mammary expression vectors for easy and faster cloning of transgenes were generated. All of these are based on a 7 kb WAP gene (clone #435 from Lothar Hennighausen). The flanking EcoR1 sites were converted into Not1 sites. The 1.6 kb Asp718 - Sal1 fragment was replaced with a polylinker containing sites for sticky and blunt cloning. The cDNA to be cloned needs to contain a start codon (AUG) and a stop codon (TAA). You do not want to have a poly(A) site (AATAAA) site in your cDNA.

Cloning:

Insert the cDNA into the polylinker site. Your inserted DNA segment needs a start (ATG) and a stop codon (e.g. TAA) but no poly(A) site (AATAAA). Excise the transgene from the vector with Not1.

All three clones have a loxP site inserted into the BamH1 site of the 3' non-translated sequence (trailer) of exon 4. Inserts at this location are of no effect on transcription of the transgene (see Burdon et al. 1991 J. Biol. Chem. 266: 6909-14).

The first vector contains only one loxP site situated in the trailer region of exon 4. It can be used for Cre mediated recombination (crosses with WAP-Cre mice) to cut out head-to-tail orientated transgens for studies of copy number dependent expression.

The second vector embodies an additional loxP sequence in the Xmn1 site (ca. 180 bp downstream of the 5' flanking Not1 site) which is in direct orientation with the loxP site in the 3' non-translated region of exon 4. The loxP sequence in the promoter region is upstream of important regulatory elements (see McKnight et al. 1995 Molecular Endocrinology 9: 717-724).

Purpose: All copies of the transgene could be removed from the genome in epithelial cells of the mammary gland at different time points (e.g. 2. lactation) with the help of the Cre-lox system to reverse the phenotype.

In contrast to the second vector, number 3 has the second loxP site between promoter and polylinker. Both loxP sites are still in direct orientation but the two sequences are inverted to avoid an additional ATG codon in front of your cDNA. The loxP itself in front of the start codon of the cDNA should not inhibit the translation of the mRNA in transgenic mice. In both experiments performed by Orban et. al. (1992) and Lakso et al. (1992) was one loxP site between promoter and coding region of active transgenes and no negative effect was reported.

The Mlu1 site in front of loxP can be used to replace the WAP promoter with other 5' regulatory elements (e.g. MMTV LTR). The WAP promoter can be cut out with Xmn1 (ca. 180 bp downstream of the 5' flanking Not1 site) and Mlu1. For this purpose, the Not1 fragment should be cloned into a vector without additional Xmn1 site (no ampicillin gene, e.g. pZEro-1 from Invitrogen).

References:
Burdon et al. (1991) J. Biol. Chem. 266: 6909-14
Lakso et al. (1992) Proc. Natl. Acad. Sci. 89: 6232-26
McKnight et al. (1995) Molecular Endocrinology 9: 717-724
Orban et al. (1992) Proc. Natl. Acad. Sci. 89: 6861-65

Note of caution:
Even if you follow our suggestions to built a mammary expression vector, the transgene may not be active for a number of reasons (we do not guarantee success). In general, the constructs used by a number of groups expressed in approximately one half of the lines generated. However, a strong variation was observed between lines. Within a line there is rather little variation.

For additional information contact
Kay-Uwe Wagner (e-mail kayuwew@bdg10.niddk.nih.gov) or
Lothar Hennighausen (e-mail mammary@nih.gov) or

last update: June 1998

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