By Philippe M Soriano, Paul Wassarman
ISBN-10: 0123848806
ISBN-13: 9780123848802
This volume comprehensively covers new technologies and methodologies that experience seemed for the learn of mouse development. This quantity is an element B of an update of quantity 225, consultant to innovations in Mouse improvement, edited via P.M. Wassarman and M.L. DePamphilis and released in 1993. Comprehensively covers new thoughts for the cryopreservation of gametes and embryos, construction of transgenic and null (knockout) animals (use of ES cells), new release of conditional/inducible mutant animals, use of gene-trap mutagenesis, research of allele-specific expression, use of latest reporter constructs, humanizing of transgenic animals, transcript profiling of mouse improvement, imaging of mouse improvement, and rederivation of animals and use of mouse genomics.
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The amount of integrated provirus per genome is determined by the means of the cycle threshold (Ct) values: DCt ¼ CtðBLR1Þ À CtðtransgeneÞ Amount provirus=genome ¼ 2DCt 5. Summary Transgenic animals play an important role in basic and clinical research. Especially, transgenesis of large mammals is necessary for studying biological functions of genes of interest and for the development of new therapies and drugs to treat human diseases (Park, 2007). Lentivirus transgenesis is a versatile tool to achieve this goal.
Transgenic Res. 13, 513–522. Pfeifer, A. (2006). Lentiviral transgenesis—A versatile tool for basic research and gene therapy. Curr. Gene Ther. 6, 535–542. , and Verma, I. M. (2001). Virus vectors and their application. In ‘‘Fields Virology,’’ (P. M. Howley, D. M. Knipe, D. Griffin, A. Martin, R. A. Lamb, B. Roizman, and S. E. ), pp. 469–491. Lippincott-Raven Publishers, Philadelphia, PA. , and Verma, I. M. (2002). Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos.
2. Perform real-time PCR according to the manufacturer’s instructions with the IQ real-time PCR system and the IQ Multiplex Powermix (BioRad) in 25 l volumes. Use primers and probes for the transgene as well as for a single-copy housekeeping gene. For the transgene, the probe should be labeled with 6-FAM at the 50 end and with Eclipse Dark Quencher (EDQ) at the 30 end. For mice the single-copy (one copy per cell/genome) housekeeping gene Burkitt lymphoma receptor 1 (BLR1) can be used to determine total DNA amount applying the specific primers 50 -CGGAGCTCAACCGAGACCT-30 (forward) and 50 -TGCAAAAGGCAGGATGAAGA-30 (reverse) and the 50 -TexasRed-labeled probe 50 -TexasRed-CTGTTCCACCTCGCAGTAGCC GAC-EDQ-30 .
Guide to techniques in mouse development: Mouse molecular genetics by Philippe M Soriano, Paul Wassarman
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