This contract will provide an expanded list of services including the following:(1) Transgenic mouse production Xenogen will perform microinjection of linearized plasmid or BAC transgene DNA provided by NIEHS investigators into pronuclei of single cell mouse embryos (FVB, B6SJL/F2, C57BL/6 or other user defined strain), and confirm integration of donor DNA into the mouse genome using polymerase chain reaction (PCR) reagents and methods provided by NIEHS investigators. If requested, NIEHS investigators may perform initial integration analyses. Xenogen will perform up to 3 DNA microinjection sessions and a minimum of 3 DNA integration positive (founder) mice will be generated for each transgenic project. Average time from receipt of construct to delivery of transgenic mice will be 12-14 weeks. (2) Conventional knockout mouse production Xenogen will perform electroporation of either 129 or B6 ES cells using targeting constructs provided by NIEHS investigators, screen electroporated ES cells for gene targeting by Southern and/or PCR analysis using methods developed by NIEHS investigators, perform blastocyst microinjection, and generate chimeras. At least 30 blastocysts will be microinjected with ES cells, transferred to uteri of pseudopregnant mice, and a minimum of 4 high percentage chimeras (2 male, 2 female) will be produced from two independent ES cell lines for each knockout project. Chimeras will be used to breed with the respective females (either B6 or 129) to determine germline transmission and, at the same time, the agouti pups will be produced on a pure 129 or B6 genetic background. The genotype of the agouti pups will be determined by Southern blot analysis and/or PCR analysis using methods developed by NIEHS investigators. Average time from receipt of construct to delivery of heterozygous mice will be 21-22 weeks. (3) Conditional knockout mouse production The targeting construct provided by NIEHS investigators will have the targeted locus flanked by loxP sequences and the Neo cassette flanked by FRT. Xenogen will electroporate either B6 or 129 ES cells with the construct and use the Neo undeleted (one round of tissue culture) or the Neo deleted (two rounds of tissue culture) targeted clones for blastocyst microinjection depending on requests from NIEHS investigators, to produce at least 4 high percentage chimeras (2 male, 2 female). Chimeras shall be used to breed with the respective females (either B6 or 129) to determine germline transmission and, at the same time, the agouti pups will be produced in a pure 129 or B6 genetic background. The genotype of the agouti pups will be determined by Southern blot analysis and/or PCR analysis using methods developed by NIEHS investigators. Average time from receipt of construct to delivery of heterozygous mice will be 21-22 weeks (one round of tissue culture) or 31-32 weeks (2 rounds of tissue culture). In FY 2008, the following projects have been initiated under this contract:GNAS-ex1GFPGNAS-ex1RedTetOITIH5tTSrtTAGlucocorticoid Receptor BetaP0-CreERT2CAG-Z-G11-EGFP Tie2-hsEH FE
