Sage Journals: Discover world-class research

Abstract

Animal cloning by nuclear transfer (NT) has made the production of transgenic animals using genetically modified donor cells possible and ensures the presence of the gene construct in the offspring. The identification of transgene insertion sites in donor cells before cloning may avoid the production of animals that carry undesirable characteristics due to positional effects. This article compares blastocyst development and competence to establish pregnancies of bovine cloned embryos reconstructed with lentivirus-mediated transgenic fibroblasts containing either random integration of a transgene (random integration group) or nuclear transfer derived transgenic fibroblasts with known transgene insertion sites submitted to recloning (recloned group). In the random integration group, eGFP-expressing bovine fetal fibroblasts were selected by fluorescence activated cell sorting (FACS) and used as nuclei donor cells for NT. In the recloned group, a fibroblast cell line derived from a transgenic cloned fetus was characterized regarding transgene insertion and submitted to recloning. The recloned group had higher blastocyst production (25.38 vs. 14.42%) and higher percentage of 30-day pregnancies (14.29 vs. 2.56%) when compared to the random integration group. Relative eGFP expression analysis in fibroblasts derived from each cloned embryo revealed more homogeneous expression in the recloned group. In conclusion, the use of cell lines recovered from transgenic fetuses after identification of the transgene integration site allowed for the production of cells and fetuses with stable transgene expression, and recloning may improve transgenic animal yields.

Get full access to this article

View all access options for this article.

References

Altschul

S.F.

, Gish

, Miller

et al. 1990. Basic local alignment search tool. J. Mol. Biol., 215:403–410.

Arat

, Gibbons

, Rzucidlo

S.J.

et al. 2002. In vitro development of bovine nuclear transfer embryos from transgenic clonal lines of adult and fetal fibroblast cells of the same genotype. Biol. Reprod., 66:1768–1774.

Arnold

D.R.

, Fortier

A.L.

, Lefebvre

et al. 2008. Placental insufficiencies in cloned animals—a workshop report. Placenta, 29,Suppl. A:S108–S110.

Baup

, Fraga

, Pernot

et al. 2009. Variegation and silencing in a lentiviral-based murine transgenic model. Transgenic Res., 19:399–414.

Beard

, Hochedlinger

, Plath

et al. 2006. Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis, 44:23–28.

Bryda

E.C.

, Pearson

, Agca

et al. 2006. Method for detection and identification of multiple chromosomal integration sites in transgenic animals created with lentivirus. Biotechniques, 41:715–719.

Cho

, Bhuiyan

M.M.

, Shin

et al. 2004. Development potential of transgenic somatic cell nuclear transfer embryos according to various factors of donor cell. J. Vet. Med. Sci., 66:1567–1573.

Clark

A.J.

, Burl

, Denning

et al. 2000. Gene targeting in livestock: a preview. Transgenic Res., 9:263–275.

de Loos

, van Vliet

, van Maurik

et al. 1989. Morphology of immature bovine oocytes. Gamete Res., 24:197–204.

10.

Dunn

D.A.

, Pinkert

C.A.

, Kooyman

D.L.

2005. Foundation review: transgenic animals and their impact on the drug discovery industry. Drug Discov. Today, 10:757–767.

11.

Fujimura

, Murakami

, Kurome

et al. 2008. Effects of recloning on the efficiency of production of alpha 1,3-galactosyltransferase knockout pigs. J. Reprod. Dev., 54:58–62.

12.

Gong

, Dai

, Fan

et al. 2004. Birth of calves expressing the enhanced green fluorescent protein after transfer of fresh or vitrified/thawed blastocysts produced by somatic cell nuclear transfer. Mol. Reprod. Dev., 69:278–288.

13.

Gordon

J.W.

, Ruddle

F.H.

1981. Integration and stable germ line transmission of genes injected into mouse pronuclei. Science, 214:1244–1246.

14.

Gossler

, Doetschman

, Korn

et al. 1986. Transgenesis by means of blastocyst-derived embryonic stem cell lines. Proc. Natl. Acad. Sci. USA, 83:9065–9069.

15.

Hammer

R.E.

, Pursel

V.G.

, Rexroad

C.E.

Jr.

et al. 1985. Production of transgenic rabbits, sheep and pigs by microinjection. Nature, 315:680–683.

16.

Hofmann

, Kessler

, Ewerling

et al. 2003. Efficient transgenesis in farm animals by lentiviral vectors. EMBO Rep., 4:1054–1060.

17.

Hofmann

, Zakhartchenko

, Weppert

et al. 2004. Generation of transgenic cattle by lentiviral gene transfer into oocytes. Biol. Reprod., 71:405–409.

18.

Houdebine

L.M.

2005. Use of transgenic animals to improve human health and animal production. Reprod. Domest. Anim., 40:269–281.

19.

Iguma

L.T.

, Lisauskas

S.F.

, Melo

E.O.

et al. 2005. Development of bovine embryos reconstructed by nuclear transfer of transfected and non-transfected adult fibroblast cells. Genet. Mol. Res., 4:55–66.

20.

Jaenisch

1988. Transgenic animals. Science, 240:1468–1474.

21.

Joshi

, Keith Pittman

, Haisch

et al. 2008. Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice. Biotechniques, 45:247–258.

22.

Keefer

C.L.

, Pant

, Blomberg

et al. 2007. Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates. Anim. Reprod. Sci., 98:147–168.

23.

Kuroiwa

, Kasinathan

, Choi

Y.J.

et al. 2002. Cloned transchromosomic calves producing human immunoglobulin. Nat. Biotechnol., 20:889–894.

24.

Laible

, Alonso-Gonzalez

2009. Gene targeting from laboratory to livestock: current status and emerging concepts. Biotechnol. J., 4:1278–1292.

25.

Lee

S.L.

, Kumar

B.M.

, Kim

J.G.

et al. 2007. Cellular composition and viability of cloned bovine embryos using exogene-transfected somatic cells. Reprod. Domest. Anim., 42:44–52.

26.

Lisauskas

S.F.

, Rech

E.L.

, Aragao

F.J.

2007. Characterization of transgene integration loci in transformed Madin Darby bovine kidney cells. Cloning Stem Cells, 9:456–460.

27.

Livak

K.J.

, Schmittgen

T.D.

2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods, 25:402–408.

28.

Lois

, Hong

E.J.

, Pease

et al. 2002. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science, 295:868–872.

29.

Miglino

M.A.

, Pereira

F.T.

, Visintin

J.A.

et al. 2007. Placentation in cloned cattle: structure and microvascular architecture. Theriogenology, 68:604–617.

30.

Miranda

, Bressan

F.F.

, Zecchin

K.G.

et al. 2009. Serum-starved apoptotic fibroblasts reduce blastocyst production but enable development to term after SCNT in cattle. Cloning Stem Cells, 11:565–573.

31.

Misra

R.P.

, Duncan

S.A.

2002. Gene targeting in the mouse: advances in introduction of transgenes into the genome by homologous recombination. Endocrine, 19:229–238.

32.

Park

2007. Lentiviral vectors: are they the future of animal transgenesis? Physiol. Genomics, 31:159–173.

33.

Prelle

, Zakhartchenko

, Wolf

2002. [Application of stem cell technology and nuclear transfer in animal models] Z. Arztl. Fortbild. Qualitatssich., 96:434–440.

34.

Roh

, Shim

, Hwang

W.S.

et al. 2000. In vitro development of green fluorescent protein (GFP) transgenic bovine embryos after nuclear transfer using different cell cycles and passages of fetal fibroblasts. Reprod. Fertil. Dev., 12:1–6.

35.

Rosenthal

, Jones

D.S.

1990. Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction. Nucleic Acids Res., 18:3095–3096.

36.

Rulicke

, Hubscher

2000. Germ line transformation of mammals by pronuclear microinjection. Exp. Physiol., 85:589–601.

37.

Sambrook

, Russell

2001. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press.

38.

Soriano

, Gridley

, Jaenisch

1987. Retroviruses and insertional mutagenesis in mice: proviral integration at the Mov 34 locus leads to early embryonic death. Genes Dev., 1:366–375.

39.

Williams

, Harker

, Ktistaki

et al. 2008. Position effect variegation and imprinting of transgenes in lymphocytes. Nucleic Acids Res., 36:2320–2329.

40.

Wolf

, Zakhartchenko

, Brem

1998. Nuclear transfer in mammals: recent developments and future perspectives. J. Biotechnol., 65:99–110.

41.

Wolf

, Schernthaner

, Zakhartchenko

et al. 2000. Transgenic technology in farm animals—progress and perspectives. Exp. Physiol., 85:615–625.

42.

, Li

, Crise

et al. 2003. Transcription start regions in the human genome are favored targets for MLV integration. Science, 300:1749–1751.

43.

Yamauchi

, Doe

, Ajduk

et al. 2007. Genomic DNA damage in mouse transgenesis. Biol. Reprod., 77:803–812.

44.

Zakhartchenko

, Mueller

, Alberio

et al. 2001. Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts. Mol. Reprod. Dev., 60:362–369.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.16 MB

Improved Production of Genetically Modified Fetuses with Homogeneous Transgene Expression After Transgene Integration Site Analysis and Recloning in Cattle

Abstract

Abstract

Get full access to this article

References

Supplementary Material