Rabbit cDNA
Rabbit heart cDNA libraries were constructed to allow for investigation of the molecular mechanisms of cardioprotection. CDNAs, rather than oligomers, were used to allow for greater specificity.
The rabbit heart model is an established research model with the advantage of being a close evolutionary reference species to humans. The use of the rabbit heart in cardiovascular research allows for investigation in a model which lacks xanthine oxidase. The rabbit heart also serves as a model similar in myosin heavy chain composition to the human as well as similar hemodynamic and homeostatic properties to the human.
cDNA Construction:
New Zealand white rabbits aged 15-20 weeks were used to construct rabbit heart cDNA libraries. Rabbit heart cDNA libraries were derived from whole heart homogenates.
Total heart RNA was isolated according to the procedure as described by Chomczynski and Sacchi (Anal Biochem 162:156-159,1987). Poly(A)+ RNA fractions were enriched by oligo(dT) cellulose chromatography and cDNA libraries were constructed using the SuperScript Plasmid System (GibcoBRL, cat. No.18248-013). First strand cDNA synthesis was performed using a Not I primer-adapter, a 44 bp single-stranded oligonucleotide containing 15 dT residues at its 3'-end linked to a Not I restriction site. The mRNA template was degraded by E. coli RNase H.
Second strand cDNA synthesis was performed using E. coli DNA Polymerase I and E. coli DNA Ligase. cDNA termini were blunt ended using T4 DNA polymerase. A double-stranded oligonucleotide containing a 4-base Sal I 5' extension and a Mlu I restriction site was added to the 5' and 3' ends of the cDNA molecules with T4 DNA ligase.
Size selection of cDNA molecules for ligation and elimination of residual adapters and low molecular weight cDNAs was performed by Sephacryl-500 column chromatography and fractions visualized by separation on a 1.4% alkaline agarose gel followed by autoradiography.
The plasmid vector pSPORT 1 (4,109 bp) was used for directional cloning. CDNAs were transformed into DH5 a competent cells. Library sizes for rabbit heart cDNA libraries were 6.2 x 10 5 , 6.8 x 10 5 , and 6.4 x 10 5.
CDNA insert size in all libraries was determined by random selection of cDNA clones from IPTG/X-gal LB-agar plates containing ampicillin followed by isolation of cDNA from recombinant bacteria by alkaline lysis using Qiagen columns and the plasmid DNA digested for 1 hour at 37 o C in a reaction mixture containing a 3-fold excess of the restriction enzyme Mlu I.
All cDNA sizes were individually confirmed by PCR at the time of microarray construction. Average insert size of cDNA clones from all rabbit heart cDNA libraries was determined to be approximately 1.6-1.8 kb.
Sequencing and sequence analysis was performed using the Applied Biosystems Taq DyeDeoxy Terminator cycle sequencing kit. After cycle sequencing and clean up the DNA samples were resolved by capillary electrophoresis on an PE BIOSYS 3700 automated sequencer. Prior to analysis of sequence data, sequence quality were evaluated using the accuracy assessment program phred of the phred/phrap/consed .
Sequence analysis was performed against the non-redundant GEN Bank/ EMBL/ DDBJ nucleotide, the non-redundant Gen Bank CDS translation/ PDB/ Swiss Prot/ PIR/ PRF peptide and the dbEST databases using the BLAST algorithm on a UNIX platform. Assignment of putative identity for cDNAs exhibiting matches to known genes required a maximum value of E=1e -25 (minimum p=10 -10 )and nucleotide sequence identity >95%.
Glycerol stocks of all cDNA's are stored at -80 degrees C.
The Rabbit cDNA table includes information on available glycerol stocks as well as putative identity information related to the gene of interest. The GenBank Number will link to the actual sequence relative to the gene of interest.
