Browse > Article
http://dx.doi.org/10.5187/JAST.2004.46.2.155

Developing a Gene-trapping Approach for Gene Identification Using Nuclear Transfer in Zebrafish  

Lee, K.Y. (Department of Animal Science, Michigan State University)
Publication Information
Journal of Animal Science and Technology / v.46, no.2, 2004 , pp. 155-164 More about this Journal
Abstract
This involves identifying and cloning trapped genes from cultured cells carrying the gene-trap constructs and generating cloned zebrafish using these cells for functional study. Gene-trapping studies in gene-trapped cells were carried out in initial and cloned zebrafish carrying gene-trap events were successfully produced based on the nuclear transplantation technique. Two kind of retroviral gene-trap constructs were adopted. The first one(SA/GFP-TP), constructed in my laboratory, carries a GFP reporter gene containing a splicing acceptor and an internal neo gene. The second one(Neo-TP), obtained from Dr. Hicks (Hicks et al., 1997), contains a promoter-less neo gene located in the LTR sequence of a retroviral vector. The infected cells were subjected to drug selection(neomycin treatment) because the two constructs carry the neomycin resistant gene. All those cells survived the neomycin treatment should carry the proviral insertions. For Neo-TP, Isolated DNA from the neomycin-resistant fibroblast cells infected by Neo-TP, was digested with EcoR1 restriction enzyme and transformed into bacteria after ligation. This procedure led to the isolation of seven clones carrying flanking cellular DNA with a typical retroviral integration signature sequence. These clones contained genomic DNA ranging from 1kb to 7kb and sequences of 300-600 bp were obtained from each of the rescued plasmids. Database searching showed that all of them share high homology to zebrafish sequences. For fish cloning using tagged cells, initially, nucleus donors directly selected from a mixture of cells(Neo-TP cells) were used. A total of 44 embryos(3.7%) out of 1179 transplants were reached blastula stage; 8 of these embryos(0.8%) hatched and 3(0.3%) of them survived to adulthood. One out of three lived cloned zebrafish has an amplified fragment and was labeled with 32P.
Keywords
Nuclear Transfer; Gene-trap; Mutagenesis; Zebrafish;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Golling. G., Amsterdam, A, Sun, Z; Antonelli, M., Maldonado, E., Chen, W., Burgess, S., Haldi, M., Artzt, K. and Farrington, S., et al. 2002. Insertional mutagenesis in zebrafish rapidly identities genes essential for early vertebrate development Nat Genet 31: 135-40.
2 Haffter, P., Granato, M., Brand, M., Mullins, M. C, Hammerschmidt, M., Kane, D. A., Odenthal, J., van Eeden, F. J., Jiang, Y J. and Heisenberg, C P. 1996. The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio, Development 123:1-36.
3 Hicks, G. G., Shi, E. G., Li, X. M., Li, C. H., Pawlak, M. and Ruley, H. E. 1997. Functional genomics in mice by tagged sequence mutagenesis, Nat Genet 16:338-44.
4 Hong, Y., Winkler, C and Schartl, M. 1998. Efficiency of cell culture derivation from blastula embryos and of chimera formation in the medaka (Oryzias latipes) depends on donor-genotype and passage number, Dev Genes Evol 208:595-602.
5 Humpherys, D., Eggan, K., Akutsu, H., Friedman, A, Hochedlinger, K., Yanagimachi, R, Lander, E. S., Golub, T. R and Jaenisch, R 2002. Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei, Proc Natl Acad Sci U. S. A. 99:12889-94.
6 Huang H, Ju B, Lee K. Y. and Lin S. 2003. Protocol for nuclear transfer in zebrafish. Cloning Stem Cells. 5(4):333-7.
7 Lai, L., Kolber Simonds, D., Park, K. W., Cheong, H. T., Greenstein, J. L., Im, G. S., Samuel, M., Bonk, A., Rieke, A. and Day, B. N., et al. 2002. Production of {alpha}-1,3-Galactosy-Itransferase Knockout Pigs by Nuclear Transfer Cloning, Science 3:3.
8 Marsh, D. J., Kum, J. B., Lunetta, K. L., Bennett, M. J., Gorlin, R J., Ahmed, S. F., Bodurtha, J., Crowe, C, Curtis, M. A and Dasouki, M., 1999. PTEN mutation spectrum and genotype- phenotype correlations in Bannayan-Riley-Ruvalcaba syndrome suggest a single entity with Cowden syndrome, Hum Mol Genet 8:1461-72.
9 McCreath, K. J., Howcroft, J., Campbell, K. H., Colman, A., Schnieke, A. E. and Kind, A J. 2000. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells, Nature 405:1066-9.
10 Penberthy, W. T., Shafizadeh, E. and Lin, S. 2002. The zebrafish as a model for human disease, Front Biosci 7:dI439-53.
11 Postlethwait, J. H. and Talbot, W. S. 1997. Zebrafish genomics: from mutants to genes, Trends Genet 13:183-90.
12 Spradling, A. C., Stem, D., Beaton, A, Rhem, E. J., Laverty, T., Mozden. N., Misra, S. and Rubin, G. M. 1999. The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes, Genetics 153:135-77.
13 Sun, L., Bradford, C. S., Ghosh, C, Collodi, P. and Barnes, D. W. 1995. ES-Iike cell cultures derived from early zebrafish embryos, Mol Mar Biol Biotechnol 4:193-9.
14 Tamashiro, K. L., Wakayama, T., Akutsu, H., Yamazaki, .Y., Lachey, J. L., Wortman, M. D., Seeley, R. J., D'Alessio, D. A., Woods, S. C., Yanagimaehi, R. and Sakai, R. R. 2002. Cloned mice have an obese phenotype not transmitted to their offspring, Nat Med 8:262-7.
15 WakamatsU, Y, Ozato, K. and Sasado, T. 1994. Establishment of a pluripotent cell line derived from a medaka(Oryzias latipes) blastula embryo, Mol Mar Biol Biotechnol 3:185-91.
16 Zon, L. I. 1999. Zebrafish: a new model for human disease, Genome Res 9:99-100.
17 Lin, S., Gaiano, N., Culp, P., Bums, J. C., Friedmann, T., Yee, J. K. and Hopkins, N. 1994. Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish, Science 265:666-9.
18 Ma, C., Fan, L., Ganassin, R, Bois, N. and Collodi, P. 2001. Production of zebrafish germ-line chimeras from embryo cell cultures, Proc Natl Acad Sci U. S. A. 98:2461-6.
19 Dooley, K. and Zon, L. I. 2000. Zebrafish: a model system for the study of human disease, Curr Opin Genet Dev 10:252-6.
20 Durick, K., Mendlein, J. and Xanthopoulos, K. G. 1999. Hunting with traps: genome-wide strategies for gene discovery and functional analysis, Genome Res 9:1019-25.
21 Gates, M. A, Kim, L, Egan, E. S., Cardozo, T., Sirotkin, H. I., Dougan, S. T., Lashkari, D., Abagyan, R., Schier, A. F. and Talbot, W. S. 1999. A genetic linkage map for zebrafish: comparative analysis and localization of genes and expressed sequences, Genome Res 9:334-47.
22 Lee, K. Y, Huang, H., Ju, B., Yang, Z. and Lin, S. 2002. Cloned zebrafish by nuclear transfer from long-term-cultured cells, Nat Biotechnol 20: 795-9.
23 Amsterdam, A., Burgess, S., Gelling, G., Chen, W., Sun, Z., Townsend, K., Farrington, S., Haldi, M. and Hopkins, N. 1999. A large-scale insertional mutagenesis screen in zebrafish, Genes Dev 13:2713-24.
24 Aparicio, S., Chapman, J., Stupka, E., Putnam, N., Chia, J. M., Dehal, P., Christoffels, A., Rash, S., Hoon, S. and Smit, A., et al. 2002. Whole-genome shotgun assembly and analysis of the genome of Fugu robripes, Science 297:1301-10.
25 Zwaal, R. R., Brooks, A., van Meurs, J., Groenen, J. T. and Plastak, R. H 1993. Target-selected gene inactivation in Caenorhabditis elegans by using a frozen trensposon insertion mutant bank, Proc Natl Acad Sci U. S. A. 90:7431-5.
26 Friedrich, G. and Soriano, P. 1993. Insertional mutagenesis by retroviruses and promoter traps in embryonic stem cells, Methods Enzymol 225: 681-701.