Canine Blastocyst cDNA Library

Contact: Michael A. Harkey, Ph.D.
Contact phone: (206) 667-3369
Contact fax: (206) 667-5978
Contact e-mail: mharkey@fredhutch.org

One product of CCEH-Cell and Molecular Reagents is an amplified cDNA library derived from Canis lupus familiaris blastocyst.

A single canine blastocyst was harvested 14 days post LH surge. Due to the uncertainty in the timing of canine fertilization, this could corresponds to a post-fertilization age of 6-10 days. The blastocyst structure was typical, with a thin expanded trophectoderm and a compact ICM constituting less than 10% of the blastocoel space.

Polyadenylated mRNA was purified with Oligo dT magnetic beads using a Miltenyi υMACS mRNA isolation kit (Cat. # 130-075-201). The entire mRNA sample was reverse transcribed with AffinityScript (Stratagene Cat. #200436). The cDNA was synthesized and amplified using reagents and primers from a Clonetech “SMART” cDNA Library Construction Kit from Clonetech (Cat. # 634901), which favors full-length cDNA. Amplified cDNA was cloned into the SfiI sites of λ TriplEX2 (Clontech) vector, using the SfiI sites embedded in the “SMART” primers, packaged as Lambda phage, and 2.5 x 106 pfu were plated, grown and harvested as a pooled library. An aliquot was thawed and titered 4/11/08 and yielded 4 x 107 pfu/ml, but this should be retested by the user.

The cDNA can be propagated as phage or excised with Cre recombinase and propagated as plasmid. Contact us for more information on the Cre vectors.

As a test of the quality of the library, full length open reading frames for KLF1, SOX2, Oct4, cMyc, and Nanog were all amplified by PCR from the library, verified by sequencing, cloned into expression vectors, and expressed as full length proteins in transfected cells.


How to Acquire Product

For extramural colleagues access to all reagents and SOPs requires execution of a Fred Hutch UBMTA and a nominal fee that covers production and mailing. Please fill out the MTA and email it to mharkey@fredhutch.org. If you need something not listed here, contact us. We may have it.

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