Sequences of Transgene Insertion Sites in Transgenic F4 Common Carp
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Transgenic Research 13: 95–96, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 95 Letter to the Editor Sequences of transgene insertion sites in transgenic F4 common carp Bo Wu ∗ , Yong-Hua Sun ∗ , Ya-Ping Wang,Yan-Wu Wang & Zuo-YanZhu ∗∗ State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China Received 2 October 2003; accepted 24 December 2003 In our previous study, fast-growing transgenic com- mon carp (Cyprinus carpio) were produced by mi- croinjection of BamHI-digested plasmid pMThGH into common carp fertilized eggs (Zhu et al., 1989). To keep the genetic diversity of transgenic group, we produced the transgenic F1 offspring by crossing one transgenic female with four transgenic males. The transgenic F4 common carp were raised by hybrid- ization between the transgenic male and transgenic female generation by generation. In F4 generation, 100% of the offspring carried pMThGH-transgene and the transgenic offspring had improved growth rate and feed utilization efficiency compared with the controls (Fu et al., 1998). However, up to the present, integra- tion information of the transgene in transgenic F4 fish has been lacking. Since firstly described by Perucho et al. (1980), the technique of plasmid rescue has been utilized in the study of various transgenic species, but it has not, to our knowledge, been employed previously in the study of transgenic fish. In this study, by use of the tech- nique of plasmid rescue, the sequences of transgene insertion sites in transgenic fish were characterized in two heaviest individuals of 1-year-old transgenic F4 fish. Total DNAs were partially digested with BamHI, gel recovered, and treated with T4 DNA ligase. The self-ligated DNA was transformed into E. Coli, and ampicillin-resistant clones were selected for plasmid preparation and analysis. Among the recovered plasmids, in spite of those having the same configuration as the original pMThGH, the other five types have aberrant config- urations, which were quite different from the orig- inal configuration. These aberrant plasmids were ∗ Bo Wu and Yong-Hua Sun made equal contributions to this work ∗∗ Author for correspondence E-mail: [email protected] considered harboring common carp genomic se- quences at insertion sites. Based on restriction analysis and Southern hybridization, host DNA fragments from three aberrant types (type I, II, III) of plasmid were subcloned into pUC19 and sequenced. We found that these three types of host DNA fragments all have high homology to the so-called ‘house-keeping’ genes of mammals or fish. In type I, a 3.2 kb fragment next to the transgene was se- quenced from both ends. The sequence of nt. 116–481 from one end was homologous to 5 regulatory se- quence of the mouse phosphoglycerate kinase-1 gene (gi|200323|gb|M18735.1|) with 99% identity (to see Genebank, accession number AF353996). The se- quences of nt. 116–481 and nt. 334–458 from the other end were homologous to mouse phosphogly- cerate kinase-1b 3 downstream regulation region in- cluding the polyA signal (gi|53670|emb|X15340.1|) with 99 and 97% identity, respectively (to see Gene- bank, accession number AF353995). This implied that the host DNA sequence of type I was the phosphoglycerate kinase-1 homologue of common carp. In type II, a 609 bp DNA fragment adja- cent to the transgene was sequenced and found 98% homologous to the upstream of human epi- dermal keratin 14 (KRT14) gene promoter region (gi|533529|gb|U11076.1|HSU11076) (to see Gene- bank, accession number AF353994). This suggested that the host DNA sequence of type II was common carp KRT14 like sequence. In type III, two DNA fragments adjacent to the transgene were analyzed. For one fragment, nt. 1–230 and nt. 241–686 were homologous to the promoter and 5 UTR region of common carp β - actin gene (gi|5881101|gb |AF170915.1|AF170915) with 100 and 99% identity, respectively. For the other fragment, a 694bp DNA sequence was found 99% homologous to common carp β -actin gene intron A
Transcript of Sequences of Transgene Insertion Sites in Transgenic F4 Common Carp
Transgenic Research 13: 95–96, 2004.© 2004 Kluwer Academic Publishers. Printed in the Netherlands.
95
Letter to the Editor
Sequences of transgene insertion sites in transgenic F4 common carp
Bo Wu∗, Yong-Hua Sun∗, Ya-Ping Wang, Yan-Wu Wang & Zuo-Yan Zhu∗∗State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy ofSciences, Wuhan 430072, China
Received 2 October 2003; accepted 24 December 2003
In our previous study, fast-growing transgenic com-mon carp (Cyprinus carpio) were produced by mi-croinjection of BamHI-digested plasmid pMThGHinto common carp fertilized eggs (Zhu et al., 1989).To keep the genetic diversity of transgenic group, weproduced the transgenic F1 offspring by crossing onetransgenic female with four transgenic males. Thetransgenic F4 common carp were raised by hybrid-ization between the transgenic male and transgenicfemale generation by generation. In F4 generation,100% of the offspring carried pMThGH-transgene andthe transgenic offspring had improved growth rate andfeed utilization efficiency compared with the controls(Fu et al., 1998). However, up to the present, integra-tion information of the transgene in transgenic F4 fishhas been lacking.
Since firstly described by Perucho et al. (1980), thetechnique of plasmid rescue has been utilized in thestudy of various transgenic species, but it has not, toour knowledge, been employed previously in the studyof transgenic fish. In this study, by use of the tech-nique of plasmid rescue, the sequences of transgeneinsertion sites in transgenic fish were characterized intwo heaviest individuals of 1-year-old transgenic F4fish. Total DNAs were partially digested with BamHI,gel recovered, and treated with T4 DNA ligase. Theself-ligated DNA was transformed into E. Coli, andampicillin-resistant clones were selected for plasmidpreparation and analysis.
Among the recovered plasmids, in spite ofthose having the same configuration as the originalpMThGH, the other five types have aberrant config-urations, which were quite different from the orig-inal configuration. These aberrant plasmids were
∗Bo Wu and Yong-Hua Sun made equal contributions to thiswork
∗∗Author for correspondenceE-mail: [email protected]
considered harboring common carp genomic se-quences at insertion sites. Based on restriction analysisand Southern hybridization, host DNA fragments fromthree aberrant types (type I, II, III) of plasmid weresubcloned into pUC19 and sequenced.
We found that these three types of host DNAfragments all have high homology to the so-called‘house-keeping’ genes of mammals or fish. In typeI, a 3.2 kb fragment next to the transgene was se-quenced from both ends. The sequence of nt. 116–481from one end was homologous to 5′ regulatory se-quence of the mouse phosphoglycerate kinase-1 gene(gi|200323|gb|M18735.1|) with 99% identity (to seeGenebank, accession number AF353996). The se-quences of nt. 116–481 and nt. 334–458 from theother end were homologous to mouse phosphogly-cerate kinase-1b 3′ downstream regulation region in-cluding the polyA signal (gi|53670|emb|X15340.1|)with 99 and 97% identity, respectively (to see Gene-bank, accession number AF353995). This impliedthat the host DNA sequence of type I was thephosphoglycerate kinase-1 homologue of commoncarp.
In type II, a 609 bp DNA fragment adja-cent to the transgene was sequenced and found98% homologous to the upstream of human epi-dermal keratin 14 (KRT14) gene promoter region(gi|533529|gb|U11076.1|HSU11076) (to see Gene-bank, accession number AF353994). This suggestedthat the host DNA sequence of type II was commoncarp KRT14 like sequence.
In type III, two DNA fragments adjacent to thetransgene were analyzed. For one fragment, nt.1–230 and nt. 241–686 were homologous to thepromoter and 5′UTR region of common carp β-actin gene (gi|5881101|gb|AF170915.1|AF170915)
with 100 and 99% identity, respectively. For the otherfragment, a 694 bp DNA sequence was found 99%homologous to common carp β-actin gene intron A
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(gi|213041|gb|M24113.1|). It is obvious that the se-quence adjacent to the transgene of A-6 was commoncarp β-actin gene.
In view of the results, it seems that the integrationevents tend to occur at the regulatory and coding se-quences in common carp genome. This interpretationis fairly reasonable, since these regions on chromo-some are regularly loosened, and the loosened DNAregions facilitate the integration of foreign gene, whilethose non-coding sequences often emerge as compactregions on chromosome. In our study, however, thetransgenic fish appeared not to suffer from any in-sertion mutagenesis. This may be due to that eachintegrated transgene was a hemizygous one, since hy-bridization between the transgenic F4 fish and thenon-transgenic ones did not generate 100% transgenicoffspring.
Acknowledgements
This work was supported by the Projects of Devel-opment Plan of the State Key Fundamental Research(G2000016109) from the Ministry of Science andTechnology, China and the National Natural ScienceFoundation of China (NSFC 90208024).
References
Fu C, Cui Y, Hung SSO and Zhu Z (1998) Growth and feed utiliza-tion by F4 human growth hormone transgenic carp fed diets withdifferent protein levels. J Fish Biol 53: 115–129.
Perucho M, Hanahan D, Lipsich L and Wigler M (1980) Isolationof the chicken thymidine kinase gene by plasmid rescue. Nature285: 207–210.
Zhu Z, Xu K, Xie Y, Li G and He L (1989) A model of transgenicfish. Sci Sin B: 147–155.
