Expressional artifact caused by a co-injection marker rol-6 in C. elegans


Autoři: HoYong Jin aff001;  Scott W. Emmons aff002;  Byunghyuk Kim aff001
Působiště autorů: Department of Life Science, Dongguk University-Seoul, Goyang, Republic of Korea aff001;  Department of Genetics and Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, United States of America aff002
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224533

Souhrn

In transgenic research, selection markers have greatly facilitated the generation of transgenic animals. A prerequisite for a suitable selection marker to be used along with a test gene of interest is that the marker should not affect the phenotype of interest in transformed animals. One of the most common selection markers used in C. elegans transgenic approaches is the rol-6 co-injection marker, which induces a behavioral roller phenotype due to a cuticle defect but is not known to have other side effects. However, we found that the rol-6 co-injection marker can cause expression of GFP in the test sequence in a male-specific interneuron called CP09. We found that the rol-6 gene sequence included in the marker plasmid is responsible for this unwanted expression. Accordingly, the use of the rol-6 co-injection marker is not recommended when researchers intend to examine precise expression or perform functional studies especially targeting male C. elegans neurons. The rol-6 sequence region we identified can be used to drive a specific expression in CP09 neuron for future research.

Klíčová slova:

3' UTR – Caenorhabditis elegans – Gene expression – Genetically modified animals – Homologous recombination – Neurons – Plasmid construction – Plasmid vectors


Zdroje

1. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science. 1994; 263: 802–805. doi: 10.1126/science.8303295 8303295

2. Altun-Gultekin Z, Andachi Y, Tsalik EL, Pilgrim D, Kohara Y, Hobert O. A regulatory cascade of three homeobox genes, ceh-10, ttx-3 and ceh-23, controls cell fate specification of a defined interneuron class in C. elegans. Development. 2001;128: 1951–1969. 11493519

3. Frøkjaer-Jensen C, Davis MW, Hopkins CE, Newman BJ, Thummel JM, Olesen SP, Grunnet M, Jorgensen EM. Single-copy insertion of transgenes in Caenorhabditis elegans. Nat Genet. 2008; 40: 1375–1383. doi: 10.1038/ng.248 18953339

4. Granato M, Schnabel H, Schnabel R. pha-1, a selectable marker for gene transfer in C. elegans. Nucleic Acids Res. 1994; 22: 1762–1763. doi: 10.1093/nar/22.9.1762 8202383

5. Praitis V, Casey E, Collar D, Austin J. Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics. 2001; 157: 1217–1226. 11238406

6. Thacker C, Sheps JA, Rose AM. Caenorhabditis elegans dpy-5 is a cuticle procollagen processed by a proprotein convertase. Cell Mol Life Sci. 2006; 63: 1193–1204. doi: 10.1007/s00018-006-6012-z 16649143

7. Kramer JM, French RP, Park EC, Johnson JJ. The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen. Mol Cell Biol. 1990; 10: 2081–2089. doi: 10.1128/mcb.10.5.2081 1970117

8. Mello CC, Kramer JM, Stinchcomb D, Ambros V. Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 1991; 10: 3959–3970. 1935914

9. Kim B, Suo B, Emmons SW. Gene Function Prediction Based on Developmental Transcriptomes of the Two Sexes in C. elegans. Cell Rep. 2016; 17: 917–928. doi: 10.1016/j.celrep.2016.09.051 27732864

10. Sulston JE, Albertson DG, Thomson JN. The Caenorhabditis elegans male: postembryonic development of nongonadal structures. Dev Biol. 1980; 78: 542–576. doi: 10.1016/0012-1606(80)90352-8 7409314

11. Loer CM, Kenyon CJ. Serotonin-deficient mutants and male mating behavior in the nematode Caenorhabditis elegans. J Neurosci. 1993; 13: 5407–5417. doi: 10.1523/JNEUROSCI.13-12-05407.1993 8254383

12. Barrios A, Ghosh R, Fang C, Emmons SW, Barr MM. PDF-1 neuropeptide signaling modulates a neural circuit for mate-searching behavior in C. elegans. Nat Neurosci. 2012; 15: 1675–1682. doi: 10.1038/nn.3253 23143519

13. Serrano-Saiz E, Pereira L, Gendrel M, Aghayeva U, Bhattacharya A, Howell K, Garcia LR, Hobert O. A Neurotransmitter Atlas of the Caenorhabditis elegans Male Nervous System Reveals Sexually Dimorphic Neurotransmitter Usage. Genetics. 2017; 206: 1251–1269. doi: 10.1534/genetics.117.202127 28684604

14. Altun ZF, Herndon LA, Wolkow CA, Crocker C, Lints R, Hall DH. WormAtlas, ed. 2002–2019; http://www.wormatlas.org

15. Ruvinsky I, Ruvkun G. Functional tests of enhancer conservation between distantly related species. Development. 2003; 130: 5133–5142. doi: 10.1242/dev.00711 12944426

16. Boulin T, Etchberger JF, Hobert O. Reporter gene fusions. WormBook, ed. The C. elegans Research Community, WormBook. 2006; doi: 10.1895/wormbook.1.106.1, http://www.wormbook.org. 18050449

17. Feinberg EH, Vanhoven MK, Bendesky A, Wang G, Fetter RD, Shen K, Bargmann CI. GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems. Neuron. 2008; 57: 353–363. doi: 10.1016/j.neuron.2007.11.030 18255029

18. Desbois M, Cook SJ, Emmons SW, Bülow HE. Directional Trans-Synaptic Labeling of Specific Neuronal Connections in Live Animals. Genetics. 2015; 200: 697–705. doi: 10.1534/genetics.115.177006 25917682

19. Okkema PG, Krause M. Transcriptional regulation. WormBook, ed. The C. elegans Research Community, WormBook. 2005; doi: 10.1895/wormbook.1.45.1, http://www.wormbook.org. 18050428

20. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974; 77: 71–94. 4366476

21. Hobert O. PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans. Biotechniques. 2002; 32: 728–730. doi: 10.2144/02324bm01 11962590

22. Kim B, Emmons SW. Multiple conserved cell adhesion protein interactions mediate neural wiring of a sensory circuit in C. elegans. Elife. 2017; 6. pii: e29257.


Článek vyšel v časopise

PLOS One


2019 Číslo 12