title: LAFUGA Cattle subtr lib 18ET v1.0
status: Public on Dec 07 2005
contact.city: Lindau
contact.first: Stefan
contact.middle: Michael
contact.last: Bauersachs
contact.email: stefan.bauersachs@uzh.ch
contact.state: Zurich
contact.address: Eschikon 27 EHB 23.1
contact.department: Department for Farm Animals
contact.country: Switzerland
contact.institute: University of Zurich
organism: Bos taurus
manufacture_protocol: Pretreatment of Animals and Collection of Endometrial Tissue Samples for the production of subtracted cDNA libraries:
  The estrous cycle of five monozygotic twin pairs (Simmental cows, non-lactating), generated by embryo splitting, which had given birth to an average of one or two calves previously, was synchronized by a single intramuscular injection of 500 µg Cloprostenol (Estrumate®; Essex Tierarznei, München, Germany) at diestrus. Animals were observed for sexual behavior (i.e. toleration, sweating, vaginal mucus) to determine standing heat, which occurred around 60 h after Estrumate® injection. Seven days after standing heat, two in vitro produced bovine blastocysts (day 7 after in vitro fertilization) in 200 µl culture medium were transferred into the ipsilateral uterine horn of one twin of each pair. In vitro production of embryos was performed as described previously (Stojkovic M. 1999) with minor modifications. The corresponding twin served as a control and received a sham-transfer of the same amount of transfer medium without embryos. On Day 18 of pregnancy, or of the estrous cycle, respectively, the animals were slaughtered. Blood samples for determination of serum progesterone levels were taken just before slaughter. All animals displayed high progesterone values (mean 3.7 ng/ml), which did not differ significantly between both groups, indicating the presence of an endocrinologically active corpus luteum. The uterine horns were opened longitudinally with a scissors and intercaruncular endometrial tissue samples from defined uterine regions (for further details see Bauersachs S. et al. 2005) were dissected using a scalpel. Pregnancy was confirmed by the presence of an apparently normal conceptus in the uterine lumen. Tissue samples of the endometrium were transferred into tubes containing 4 ml of RNAlater™ (Ambion, Huntigton, UK) within 20 minutes after slaughter, stored overnight at 4°C and then at -20°C until further processing. For gene expression analyses only those tissues samples of pregnant animals, which had been in contact with an embryo were used, i.e. the middle and caudal sections of the uterine horn ipsilateral to the ovary bearing the corpus luteum. From control animals, endometrial samples from the same uterine sections were analyzed. All experiments with animals were carried out with permission from the local veterinary authorities.
  Generation of Subtracted cDNA Libraries:
  One twin pair was selected for the preparation of a subtracted library enriched for cDNAs of genes up-regulated due to the presence of a conceptus during the pre-implantation period. Total RNA was isolated using Trizol™ reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer's recommendations. Quality of total RNA was checked by agarose gel electrophoresis and quantity was determined by spectrometry. The first subtracted library was generated using the Suppression Subtractive Hybridization (SSH) method (Diatchenko et al. 1996) and was done as reported previously (Bauersachs S. et al. 2003; Bauersachs S. et al. 2004) with a minor modification: at the second step of subtractive hybridization no driver cDNA was added. A second library was constructed using a purchased subtracted cDNA (vertis AG, Freising, Germany). Subtraction was performed as previously described (Ros B. et al 2004) using the same RNA samples as used for construction of the SSH library.
  Preparation of cDNA arrays:
  4,608 cDNA clones were randomly picked from the two subtracted libraries (SSH library: 3072, vertis library: 1536) and grown overnight at 37 °C in 100 µl LB medium in 96-well microtiter plates. Bacterial suspensions were diluted 20-fold in TE buffer (pH 8.0), incubated at 96 °C for 10 min, and stored at -20 °C. PCR amplification of the cDNA insertions was performed in 20 µl in 96-well cycle plates and contained 1 µl diluted bacterial cells, PCR buffer (peQLab, Erlangen, Germany), 200 pmol/µl dNTPs, 0.3 pmol/µl of the respective primers (modified T3 and T7 primers), and 1 U Taq DNA polymerase (peQLab). All PCR products were analyzed by 0.8 % agarose gel electrophoresis showing that more than 95 % of the PCR reactions yielded defined PCR products. 15 µl of PCR reactions were transferred to 384-well microtiter plates (ABgene) containing 15 µl 2-fold spotting buffer (40 mmol/l Tris-HCl pH 8, 2 M NaCl, 2 mmol/l EDTA, bromophenol blue). 1,536 PCR products were spotted onto nylon membranes (Nytran Supercharge, Schleicher & Schuell, Dassel Germany) on an area of 20 x 50 mm using an Omnigrid Accent microarrayer (GeneMachines, San Carlos, California, USA) and solid pins (SSP015, diameter 0.015 inches; Telechem International, Sunnyvale, California, USA). Spotting was done 6 times for each PCR product on the same position for sufficient and equal application. 25 arrays were produced simultaneously. The cDNAs were denatured on the arrays by incubation on filter paper (Schleicher & Schuell) soaked with 0.5 N NaOH for 20 min at room temperature. DNA was fixed by baking at 80 °C for 30 min and UV cross-linking (120 mJ/cm2, XL-1500 UV Crosslinker, Spectronics Corp., New York, USA). A set of three arrays were produced (A, B, C) whereas arrays A and B contained the cDNA clones of the SSH library and array C contained the clones of the vertis library.