Activity and Global Carbon Cycle Changes over the Past 50,000 Years

27. L. Redmond, S.-R. Oh, C. Hicks, G. Weinmaster, A. Ghosh, Nature Neurosci. 3, 30 (2000). 28. To create the CREST antibody, We used PCR to amplify full-length crest cDNA from P0 mouse brain total RNA and cloned it into pET21 vector for expression of His-tagged CREST protein in bacteria. After isopropyl-D-thiogalactopyranoside induction, CREST protein was mainly collected in the inclusion body of the bacteria and extracted with 6 M urea. We further purified the protein with Ni-NTA agarose fractionation. After SDS–polyacrylamide gel electrophoresis (PAGE), the Coomassie brilliant blue– stained CREST band was excised and injected into rabbits to produce anti-CREST antiserum. 29. To generate the crest knockout mice, crest genomic DNA was cloned by screening a 129SVJ mouse genomic phage library (gift of A. Kolodkin) with full-length mouse crest cDNA as a probe. After mapping the restriction enzyme sites, we generated a knockout vector with a Neo gene cassette (Fig. 4). We deleted the poly(A) addition signal from the Neo gene cassette for poly(A) trapping method of gene targeting. The cassette replaced all of exon 4 and a 5 portion of exon 5. ES cells transfected with the targeting vector and resistant to G418 and gancyclovir were expanded and screened by genomic Southern blotting. Correctly targeted ES cells were injected into C57B6J-derived blastocysts and resulted in the generation of several high-percentage chimeras, which produced germline targeted offspring. Genotyping of mice was performed on tail clip DNA by PCR. 30. We thank D. Livingston for the UAS-CAT construct; A. Lanahan for advice on library construction; A. Kolodkin for rat phage libraries; L. Redmond, A. Datwani, K. Whitford, M.-R. Song, and G. Ince for various procedures; J. Nathans, C. Montell, D. Ginty, P. Worley, S. Snyder, D. Linden, D. Murphy, P. Kim, M. Molliver for discussions; and M. Greenberg, D. Ginty, A. Kolodkin, and S. Snyder for comments on the manuscript. Supported by grants from NIH (MH60598 and NS39993), the March of Dimes Birth Defects Foundation (A.G.), the Klingenstein Foundation (A.G.), and a Merck Scholar Award (A.G.). H.A. was supported by a Uehara Memorial Foundation Research Fellowship.