Rapid in vivo functional analysis of transgenes in mice using whole body imaging of luciferase expression

Transgenic Research - 2001
Weisheng Zhang1, Jian Q. Feng2, Stephen E. Harris3, Pamela R. Contag4, David K. Stevenson1, Christopher H. Contag1
1Division of Neonatal and Developmental Medicine, Departments of Pediatrics, and Microbiology and Immunology, Stanford University Medical Center, Stanford University, Stanford
2Department of Oral Biology, University of Missouri-Kansas City, Kansas City
3Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio
4Xenogen Corporation, Alameda

Tóm tắt

The use of transgenic animals in biomedical research is increasing rapidly and may be the best means of determining gene function. Generating transgenic animals typically requires time-consuming screening processes, and gene function is assessed by an array of difficult phenotypic and biochemical assays performed ex vivo. To address the unmet need in transgenic research for functional assays performed with ease in living animals, we demonstrate here that in vivo detection of luciferase enzyme as a transcriptional reporter facilitates rapid screening for both the presence and function of transgenes in intact living mice. Using this approach we identified three bioluminescent transgenic founders where the transgene consisted of the heme oxygenase promoter fused to the modified coding sequence of the luciferase gene. These founders were identified from 183 pups and confirmed by PCR analysis. Identification of HO-1-luc homozygotes from back-crossed F2 littermates was then accelerated by in vivo imaging. In another transgenic mouse line, where the transgene was comprised of the bone morphogenic-4 (BMP4) promoter fused to the modified luciferase gene, we were able to identify transgenic animals and in each line we were able to visualize patterns of expression in living animals over time. The light production from these transgenic mice indicated that the desired DNA fragment was functional and different expression profiles apparent at different ages and after gene induction.

Từ khóa


Tài liệu tham khảo

Alam J (1994) Multiple elements within the 5' distal enhancer of the mouse heme oxygenase-1 gene mediate induction by heavy metals. J Biol Chem 269(40): 25049–25056.

Bergeron M, Ferriero DM and Sharp FR (1998) Developmental expression of heme oxygenase-1 (HSP32) in rat brain: an immunocytochemical study. Brain Res Dev Brain Res 105: 181–194.

Bieberich CJ, King CM, Tinkle BT and G Jay (1993) A transgenic model of transactivation by the tax protein of HTLV-I. Virol 196: 309–318.

Bieberich CJ, Ngo L and Jay G (1994) Molecular approaches involved in mammalian gene transfer: Evaluation of transgene expression. In: Pinkert CA (ed.), Transgenic Animal Technology: A Laboratory Handbook. pp. 235–262. Academic Press, San Diedo, California.

Brandes C, Plautz JD, Stanewsky R, Jamison CF, Straume M, Wood KV et al. (1996) Novel features of Drosophila period transcription revealed by real-time luciferase reporting. Neuron 16: 687–692.

Campbell AK (1988) Chemiluminescence. Principles and applications in biology and medicine. Ellis Horwood Ltd. and VCH Verlagsgesellschaft mbH, Chichester, England.

Chance B, Luo Q, Nioka S, Alsop DC, Detre JA (1977) Optical investigations of physiology: a study of intrinsic and extrinsic biomedical contrast. Philos Trans R Soc Lond B Biol Sci 352: 707–716.

Contag PR, Olomu I, Stevenson DK and Contag CH (1998) Bioluminescent reporters in living mammals. Nature Med 4: 245–247.

Contag CH, Spilman SD, Contag PR, Oshiro M, Eames B, Dennery P et al. (1997) Visualizing gene expression in living mammals using a bioluminescent reporter. Photochem Photobiol 66: 523–531.

Contag CH, Contag PR, Mullins JI, Spilman SD, Stevenson DK and Benaron DA (1995) Photonic detection of bacterial pathogens in living hosts. Mol Microbiol 18: 593–603.

Contag CH, Jenkins D, Contag PR and Negrin RS (2000) Use of reporter genes for optical measurements of neoplastic disease in vivo. Neoplasia 2: 41–52.

Edinger M, Sweeney TJ, Tucker AA, Olomu AB, Negrin RS and Contag CH (1999) Noninvasive assessment of tumor cell proliferation in animal models. Neoplasia 1: 303–310.

Feng JQ, Harris MA, Ghosh-Choudhury N, Feng M, Mundy GR, Harris SE (1994) Structure and sequence of mouse bone morphogenetic protein-2 gene (BMP-2): comparison of the structures and promoter regions of BMP-2 and BMP-4 genes. Biochimica et Biophysica Acta 1218: 221–224.

Hammerschmidt M, Serbedzija GN, McMahon AP (1996) Genetic analysis of dorsoventral pattern formation in the zebrafish: requirement of a BMP-like ventralizing activity and its dorsal repressor. Genes Dev 10: 2452–2461.

Hastings JW (1996) Chemistries and colors of bioluminescent reactions: a review. Gene 173: 5–11.

Johnson J, Wold B and Hauschka S (1989) Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice. Mol Cell Biol 9: 3393–3399.

Liem KF Jr, Tremml G, Roelink H and Jessell TM (1995) Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. Cell 82(6): 969–979.

Lipshutz G, Gruber C, Cao Y-A, Hardy J, Contag C and Gaensler K (2001) Utero delivery of adeno-associated viral vectors: Intraperitoneal gene transfer produces long-term expression. Mol Ther (in press).

Liu ML, Olson AL, Moye-Rowley WS, Buse JB, Bell GI and Pessin JE (1992) Expression and regulation of the human GLUT4/muscle-fat facilitative glucose transporter gene in transgenic mice. J Biol Chem 267: 11673–11676.

Kim J, Ault KT, Chen HD, Xu RH, Roh DH, Lin MC et al. (1998). Transcriptional regulation of BMP-4 in the Xenopus embryo: analysis of genomic BMP-4 and its promoter. Biochem Biophys Res Commun 250(2): 516–530.

Millar AJ, Short SR, Chua NH and Kay SA (1992) A novel circadian phenotype based on firefly luciferase expression in transgenic plants. Plant Cell 4: 1075–1087.

Monastersky G Mand Robl JM (ed.) (1995) Strategies in Transgenic Animal Science. ASM Press, Washington D.C.

Morrey JD, Bourn SM, Bunch TD, Thomas D, Siddwell RW and Rosen CA (1992) HIV-1 LTR activation model: Evaluation of various agents in skin of transgenic mice. J Acquir Immune Defic Syndr 5: 1195–1203.

Morrey JD, Bourn SM, Bunch TD, Jackson MK, Siddwell RW, Barrows LR et al. (1991) In vivo activation of human immuno-deficiency virus type 1 long terminal repeat by UV type A (UV-A) light plus psoralen and UV-B light in the skin of transgenic mice. J Viol 65: 5045–5051.

Rindt H, Gulick J, Knotts S, Neumann J and Robbins J (1993) In vivo analysis of the murine beta-myosin heavy chain gene promoter. J Biol Chem 268: 5332–5338.

Pinkert CA (ed.) (1994) Transgenic Animal Technology: A Laboratory Handbook. Academic Press, San Diedo, California.

Rehemtulla A, Stegman L, Cardozo S, Gupta S, Hall D, Contag C et al. (2000). Rapid and quantitative assessment of cancer treatment response using in vivo bioluminescence imaging. Neoplasia 2: 491–495.

Rodgers PA and Stevenson DK (1990) Developmental biology of heme oxygenase. Clin Perinatol 17(2): 275–291.

Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual (2nd edn). Cold Spring Harbor Laboratory Press, USA.

Seidman CE, Schmidt EV and Seidman JG (1991) Cis-dominance of rat atrial natriuretic factor gene regulatory sequences in transgenic mice. Can J Physiol Pharmacol 69: 1486–1492.

Stanewsky R, Jamison CF, Plautz JD, Kay SA and Hall JC (1997) Multiple circadian-regulated elements contribute to cycling period gene expression in Drosophila. EMBO J 16: 5006–5018.

Tinkle BT, Bieberich CJ and Jay G (1994) Molecular approaches involved in mammalian gene transfer: Analysis of transgene integration. In: Pinkert CA (ed.), Transgenic Animal Technology: A Laboratory Handbook. (pp. 221–234). Academic Press, San Diedo, California.

Tsika RW, Bahl JJ, Leinwand LA and Morkin E (1990) Thyroid hormone regulates expression of a transfected human alpha-myosin heavy chain fusion gene in fetal rat heart cells. Proc Natl Acad Sci USA 87: 379–383.

Walsh A, Azrolan N, Wang K, Marcigliano A, O'Connell A and Breslow JL (1993) Intestinal expression of the human apo A-I gene in transgenic mice is controlled by a DNA region 3' to the gene in the promoter of adjacent convergently transcribed apo C-II gene. J Lipid Res 34: 617–623.

Walsh A, Ito Y and Breslow JL (1989) High levels of human apolipoprotein A-I in transgenic mice result in increased plasma levels of small high density lipoprotein (HDL) particles comparable to human HDL3. J Biol Chem 264: 6488–6494.

Watanabe Y and Le Douarin NM (1996) A role for BMP-4 in the development of subcutaneous cartilage. Mech Dev 57(1): 69–78.

Weng Y-H, Tatarov A, Bartos BP, Contag CH and Dennery PA (2000) Bioluminescent and doxycycline-regulatable HO-1 expression in type II pneumocytes via transpulmonary delivery. Am J Physiol Lung Cell Mol Physiol 278: L1273–1279.

Wozney J, Mosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz MJ et al. (1988) Novel regulators of bone formation: molecular clones and activities. Science 242: 1528–1534.

Thông tin
Thông tin xuất bản

Transgenic Research

Thông tin tác giả