Molecular Markers for Mammary Differentiation (MMD)

Accumulation of milk protein mRNAs in mouse mammary tissue during pregnancy is differentially regulated. For example, high levels of WDNM1 RNA can be found already in early pregnancy, beta-casein accumulates at mid-pregnancy, WAP between day 14 and 16 of pregnancy and alpha-lactalbumin around parturition (see Robinson et al., 1995). The temporal activation of these genes most likely represents different stages of mammary development and differentiation.

Probes are available for the following differentiation markers:

• WAP
  WAP RNA can easily be detected with an antisense oligonucleotide
  (5' CAA CGC ATG GTA CCG GTG TCA 3')
  which spans the first AUG. For those who do not wish to use oligonucleotides, a WAP cDNA clone is available (Hennighausen and Sippel, 1982a). A 450 bp WAP cDNA can be excised from a pBS- vector (Stratagene) with PstI.
• WDNM1
  WDNM1 RNA can easily be detected with an antisense oligonucleotide
  (5' CAG AGC CCA GGC AGT AGT CAT TGT C 3')
• beta-casein
  beta-casein RNA can easily be detected with an antisense oligonucleotide
  (5' GTC TCT CTT GCA AGA GCA AGG GCC 3')

Hybridization conditions for northern blots

• incubate for 1 hr at 55C in 0.4M NaCl, 1% SDS, 1x Denhardt, salmon sperm DNA
• incubate for 6 hours at 55C in 0.4M NaCl, 1% SDS, 1x Denhardt, salmon sperm DNA, and 1ng of gamma P-32 labelled oligonucleotide probe
• wash twice for 15 min. in 1x SSC, 0.1% SDS
• wash twice for 15 min. in .2x SSC, 0.1% SDS

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  RT-PCR assay for WAP RNA

  RT primer
  5' TAG CAG CAG ATT GAA AGC ATT ATG 3'

  PCR primer
  5' GAC ACC GGT ACC ATG CGT TG 3' reannealing temp.: 60C
  25 cycles

  the product should have a size of 500 nt. Since the primers come from the 4th and 1st exon, respectively, you should not see any amplified genomic DNA.

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  For additional information contact
  Lothar Hennighausen (e-mail mammary@nih.gov)
  

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  last update: June 1998

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