Assessment of Fecundity and Germ Line Transmission in Two Transgenic Pig Lines Produced by Sleeping Beauty Transposition

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

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Distribution, expression and germ line transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs

Development ◽

10.1242/dev.99.1.15 ◽

1987 ◽

Vol 99 (1) ◽

pp. 15-23

Author(s):

L.D. Etkin ◽

B. Pearman

Keyword(s):

Xenopus Laevis ◽

Dna Sequences ◽

Copy Number ◽

Germ Line ◽

Gene Promoter ◽

Early Gene ◽

Mosaic Pattern ◽

Exogenous Dna ◽

Gene Coding ◽

Germ Line Transmission

We analysed the fate, expression and germ line transmission of exogenous DNA which was microinjected into fertilized eggs of Xenopus laevis. DNA was injected into fertilized eggs within 1 h following fertilization. The injected DNA was dispersed around the site of injection and became localized to cleavage nuclei by stage 6. Injected DNA persisted in the tissues of 6- to 8-month-old frogs and exhibited a mosaic pattern of distribution with regard to the presence or absence and copy number between different tissues. We detected the exogenous DNA sequences in 60% of injected frogs. Restriction digestion analysis of this DNA suggested that it is not rearranged and was organized as head-to-tail multimers. The copy number varied from 2 to 30 copies/cell in various tissues of the same frog. Plasmid pSV2CAT which contains the prokaryotic gene coding for chloramphenicol acetyl transferase (CAT) enzyme linked to the SV40 early gene promoter was expressed in 50% of the animals containing the gene. The pattern of expression, however, varied between different animals and could not be correlated with copy number. We also showed that the exogenous DNA sequences were transmitted through the male germ line and that each offspring contained the gene integrated into a different region of the genome.

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No evidence for germ-line transmission following prenatal and early postnatal AAV-mediated gene delivery

The Journal of Gene Medicine ◽

10.1002/jgm.718 ◽

2005 ◽

Vol 7 (5) ◽

pp. 630-637 ◽

Cited By ~ 14

Author(s):

Marcus Jakob ◽

Christiane Mühle ◽

Jung Park ◽

Susi Weiß ◽

Simon Waddington ◽

...

Keyword(s):

Gene Delivery ◽

Germ Line ◽

Germ Line Transmission

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No evidence of germ-line transmission by adenovirus-mediated gene transfer to mouse testes

Fertility and Sterility ◽

10.1016/j.fertnstert.2007.04.062 ◽

2008 ◽

Vol 89 (5) ◽

pp. 1448-1454 ◽

Cited By ~ 12

Author(s):

Yoshiyuki Kojima ◽

Yutaro Hayashi ◽

Satoshi Kurokawa ◽

Kentaro Mizuno ◽

Shoichi Sasaki ◽

...

Keyword(s):

Gene Transfer ◽

Germ Line ◽

Germ Line Transmission

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Germ-line transmission of a disrupted β2-microglobulin gene produced by homologous recombination in embryonic stem cells

Trends in Genetics ◽

10.1016/0168-9525(90)90036-6 ◽

1990 ◽

Vol 6 ◽

pp. 6 ◽

Cited By ~ 1

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Homologous Recombination ◽

Germ Line ◽

Embryonic Stem ◽

Β2 Microglobulin ◽

Germ Line Transmission

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Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission

Developmental Dynamics ◽

10.1002/(sici)1097-0177(199705)209:1<85::aid-aja8>3.0.co;2-t ◽

1997 ◽

Vol 209 (1) ◽

pp. 85-91 ◽

Cited By ~ 125

Author(s):

Xin Liu ◽

Hong Wu ◽

Janet Loring ◽

Sheriar Hormuzdi ◽

Christine M. Disteche ◽

...

Keyword(s):

Gene Targeting ◽

Potential Problem ◽

Germ Line ◽

Es Cells ◽

Germ Line Transmission

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Attempts towards derivation and establishment of bovine embryonic stem cell-like cultures

Reproduction Fertility and Development ◽

10.1071/rd04117 ◽

2005 ◽

Vol 17 (2) ◽

pp. 113 ◽

Cited By ~ 23

Author(s):

Poul Maddox-Hyttel ◽

Jakob O. Gjørret

Keyword(s):

Stem Cells ◽

Adult Stem Cells ◽

Germ Line ◽

Current Knowledge ◽

Mammalian Species ◽

Embryonic Stem ◽

Transgenic Animals ◽

Great Expectations ◽

Safety Issues ◽

Germ Line Transmission

Current knowledge on the biology of mammalian embryonic stem cells (ESC) is stunningly sparse in light of their potential value in studies of development, functional genomics, generation of transgenic animals and human medicine. Despite many efforts to derive ESC from other mammalian species, ESC that retain their capacity for germ line transmission have only been verified in the mouse. However, the criterion of germ line transmission may not need to be fulfilled for exploitation of other abilities of these cells. Promising results with human ESC-like cells and adult stem cells have nourished great expectations for their potential use in regenerative medicine. However, such an application is far from reality and substantial research is required to elucidate aspects of the basic biology of pluripotent cells, as well as safety issues associated with the use of such cells in therapy. In this context, methods for the derivation, propagation and differentiation of ESC-like cultures from domestic animals would be highly desirable as biologically relevant models. Here, we review previously published efforts to establish bovine ESC-like cells and describe a procedure used in attempts to derive similar cells from bovine Day 12 embryos.

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90 GERM-LINE TRANSMISSION OF DONOR MITOCHONDRIAL DNA IN NUCLEAR TRANSFER-DERIVED COWS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab90 ◽

2007 ◽

Vol 19 (1) ◽

pp. 162

Author(s):

K. Takeda ◽

K. Kaneyama ◽

M. Tasai ◽

S. Akagi ◽

M. Yonai ◽

...

Keyword(s):

Mitochondrial Dna ◽

Nuclear Transfer ◽

Germ Line ◽

Donor Cell ◽

Cumulus Cells ◽

Single Strand Conformation Polymorphism ◽

Loop Region ◽

D Loop ◽

Germ Line Transmission ◽

Host Embryo

In embryos derived by nuclear transfer (NT), fusion, or injection of donor cells with recipient oocytes caused mitochondrial heteroplasmy. Previous studies have reported varying patterns of mitochondrial DNA (mtDNA) transmission in cloned calves. Distribution of donor mtDNA found in offspring of NT-derived founders may also vary from donor–host embryo heteroplasmy to host embryo homoplasmy. Here we examined the transmission of mtDNA from NT cows to their progeny. NT cows were originally produced by fusion of enucleated oocytes with Jersey (J) or Holstein (H1) oviduct epithelial cells, or Holstein (H2) or Japanese Black (B) cumulus cells, as previously reported (Goto et al. 1999 Anim. Sci. J. 70, 243–245; Yonai et al. 2005 J. Dairy Sci. 88, 4097–4110; Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). Transmission of donor cell mtDNA was analyzed by PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis of the mitochondrial D-loop region. Eleven NT founder cows were analyzed, 4 (2 = J-NT, and 2 = H1-NT) of them were heteroplasmic whereas 7 (1 = J-NT, 1 = H1-NT, 2 = H2-NT, and 3 = B-NT) were homoplasmic for the host embryo mitochondria. The proportions of donor mtDNA detected in one J-NT cow was 7.7%, and those of other cow lineages were <2%. Heteroplasmic NT cows delivered a total of 9 progeny. Four of the 9 progeny exhibited heteroplasmy with high percentages of donor cell mtDNA populations (52%, 37%, 17%, and 43%). The other 5 progeny were obtained from heteroplasmic NT cows, and all samples of the 10 progeny obtained from the homoplasmic NT cows did not harbor detectable donor cell mtDNA. A genetic bottleneck in the female germ-line will generally favor the transmission of a single mitochondrial population, leading to a return to homoplasmy. Thus, some of progeny maintained heteroplasmy with a higher ratio than that of their NT mothers, which may also reflect a segregation distortion caused by the proposed mitochondrial bottleneck. These results demonstrated that donor mtDNA in NT cows could be transmitted to progeny with varying efficiencies, in a lineage-specific fashion.

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329 FLOW CYTOMETRIC ANALYSIS OF SPERMATOZOA FROM REPORTER TRANSGENIC BOARS DERIVED BY PRECISION GENETIC ENGINEERING

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab329 ◽

2013 ◽

Vol 25 (1) ◽

pp. 312

Author(s):

W. Garrels ◽

S. Holler ◽

N. Cleve ◽

S. Klein ◽

Z. Ivics ◽

...

Keyword(s):

Germ Line ◽

Flow Cytometric Analysis ◽

Embryonic Stem ◽

Sleeping Beauty ◽

Large Animal Model ◽

Large Animal ◽

Reporter Protein ◽

Cassette Exchange ◽

Flow Cytometric

Recently, we produced 2 founder boars with a non-autonomous Sleeping Beauty (SB) system carrying 3 monomeric integrations of a Venus transposon cassette and showing transgene segregation during meiosis (Garrels et al. 2011 PLoS One 6, e27563). It was possible to show transmission of the reporter protein to fertilized oocytes by confocal microscopy. The aim of this study was to assess the suitability of different fluorophore reporters for in vivo labelling of pig spermatozoa. Therefore, we used Venus transposon fibroblasts from a F1 boar, which carry a single integration of the transposon cassette and used these fibroblasts for a Cre-mediated cassette exchange against an mCherry reporter. These cells were used for somatic cell nuclear transfer (SCNT) to derive a syngene clone cohort of boars, which differ only in the fluorophore reporter cDNAs (either Venus or mCherry). Importantly, this methodology did not request any antibiotic selection cassette and allows precise genetic modifications in a livestock species where no authentic embryonic stem cells are available (Garrels et al. 2012 Trends in Biotechnology 30, 386–393). A total of 8 male piglets carrying the Venus transposon, and 4 male piglets carrying the mCherry reporter were born. Three Venus boars and 2 mCherry boars were raised to sexual maturity, and ejaculated sperm was obtained with the help of a phantom. A detailed flow cytometric analysis revealed that the spermatozoa samples were specifically Venus or mCherry positive [Gallios, Beckmann Coulter, Krefeld, Germany; solid-state laser (488 nm; 22 mW), filter for green fluorescence (525 BP); filter for red fluorescence: (620/30)], respectively. In direct comparative measurements, the spermatozoa samples from transgenic boars (Venus and Cherry) and wildtype controls could be discriminated. Interestingly, spermatozoa were uniformly Venus- or mCherry-positive and gave a distinct fluorescence peak in flow-cytometric measurements. The monomeric transgenes were transmitted through the germ line according to Mendelian rules with the expected ratio of 50% transgenic and 50% nontransgenic piglets. Fluorescence microscopic analysis and Western blotting confirmed the uniform presence of Venus and mCherry in boar spermatozoa, respectively. This is the first characterisation of spermatozoa from a pig cohort carrying a targeted cassette exchange. This large animal model may help to elucidate the function of paternally transmitted components to fertilized oocytes.

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