Transgene detection during early murine embryonic development after pronuclear microinjection

Page, Raymond; Canseco, R. S.; Russell, Christopher G.; Johnson, John L.; Velander, William H.; Gwazdauskas, F.C.

doi:10.1007/bf01976496

Cited by 16 publications

(8 citation statements)

References 25 publications

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“…Both groups were co-injected with the intramolecular-integration assay vector pBCPB [1] carrying attB and attP sites. The embryos were frozen at the two-cell stage, total DNA was extracted [42], and the expected wild-type attL junction was PCR amplified from recombined plasmid. A product of correct size was observed in both groups of embryos.…”

Section: Resultsmentioning

confidence: 99%

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Phage integrases for the construction and manipulation of transgenic mammals

Hollis

Stoll

Sclimenti

et al. 2003

Reprod Biol Endocrinol

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Phage integrases catalyze site-specific, unidirectional recombination between two short att recognition sites. Recombination results in integration when the att sites are present on two different DNA molecules and deletion or inversion when the att sites are on the same molecule. Here we demonstrate the ability of the φC31 integrase to integrate DNA into endogenous sequences in the mouse genome following microinjection of donor plasmid and integrase mRNA into mouse single-cell embryos. Transgenic early embryos and a mid-gestation mouse are reported. We also demonstrate the ability of the φC31, R4, and TP901-1 phage integrases to recombine two introduced att sites on the same chromosome in human cells, resulting in deletion of the intervening material. We compare the frequencies of mammalian chromosomal deletion catalyzed by these three integrases in different chromosomal locations. The results reviewed here introduce these bacteriophage integrases as tools for site-specific modification of the genome for the creation and manipulation of transgenic mammals.

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Section: Resultsmentioning

confidence: 99%

“…Standard embryo DNA retrieval technique for PCR was employed [42]. The primers attBF2 (ATG TAG GTC ACG GTC TCG AAG C) and attP1+ (TGG CGG CCG CTC TAG AAC TA) were used to specifically amplify the wild type attL junction in integrase-reacted pBCPB.…”

Section: Methodsmentioning

confidence: 99%

Phage integrases for the construction and manipulation of transgenic mammals

Hollis

Stoll

Sclimenti

et al. 2003

Reprod Biol Endocrinol

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“…Brinster et al [1] reported it was the pronuclear microinjection of DNA that hindered maturation of embryos, not the injection process. Furthermore, loss of injected zygotes occurs during earliest embryo development [14]. Schmotzer et al [12] reported similar findings for cytoplasmic injection of DNA.…”

Section: Discussionmentioning

confidence: 63%

“…The opposite is also true. When PCR is applied to embryo biopsies for the purpose of screening positive embryos for transplant, as in the case of mosaic embryos, it is possible to miss a positive embryo if it expresses the transgene in a mosaic fashion [14]. Furthermore, they noted that PCR of embryos is not sensitive enough to distinguish integrated from non-integrated DNA.…”

Section: Discussionmentioning

confidence: 99%

Murine embryo development following cytoplasmic injection of linear and condensed DNA

Dunlap-Brown¹,

Butler²,

Velander³

et al. 2012

OJAS

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In 1985 Brinster et al.[1] observed that linearized DNA injected in the cytoplasm of mouse zygotes underwent spontaneous supercoiling within 24 h. This finding suggests that DNA prefers and functions best in tertiary structure. In an effort to improve the efficiency of transgenesis by cytoplasmic injection, DNA was condensed with MgCl 2 to form a three dimensional rod-shaped DNA prior to injection in pronuclear stage murine zygotes. The DNA used was enhanced green fluorescent protein on a cytomegalovirus promoter (CMV-EGFP) and served as a marker for gene integration and protein expression in culture conditions. The condensed CMV-EGFP construct was injected in the cytoplasm at 3 concentrations (100, n = 816; 425, n = 464; and 625 µg/ml, n = 708). For comparison linear CMV-EGFP construct was injected into the pronucleus (5 µg/ml, n = 196) and into the cytoplasm (625 µg/ml, n = 628). In all treatment groups the control and buffer injected embryos developed similarly. Among DNA treatment groups, the highest development of fluorescing embryos was observed in zygotes injected in the cytoplasm with linear CMV-EGFP (625 µg/ml); however, zygotes injected in the cytoplasm with condensed CMV-EGFP (625 µg/ml) had the highest percentage (44%) of fluorescing embryos, the highest percentage (16.7%) of fluorescing morula and blastocysts, and the lowest percentage of fluorescence mosaicism at every stage of embryo development after 4 d in culture; thereby making it the best method for generating transgenic animals.

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“…Recently it has been shown the inbred strain FVB/N (//-2 q) is comparable to the hybrid strain, B6SJL, in many aspects of transgene production with the added advantage that the former provides a fixed genetic background (Taketo et al, 1991). In some instances an outbred strain, such as CD-1, has been used for receiving the transgene (Page et al, 1995). Upon amplification of the CD-1 DNA with H-2 specific primers, three bands were observed, one of which corresponded to the H-2 q allele (results not shown).…”

Section: Discussionmentioning

confidence: 99%

TrackingH-2 alleles in transgenic mice by RFLP and heteroduplex analysis

Shanmugam

Haines

Lake

et al. 1996

Transgenic Research

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Transgenic mice provide valuable tools for biological research including many areas of immunology. In studies involving the major histocompatibility complex (MHC), it is often necessary to place the desired transgene in a specific H-2 (the murine MHC) environment. In this regard, the strains commonly used for the production of transgenic mice also carry well characterized H-2 alleles and provide an appropriate genetic background for MHC related experiments. In this study, a highly polymorphic microsatellite of tetranucleotide repeats from the second intron of the class II Eb gene within the H-2 complex was used in order to identify the corresponding alleles. The relevant H-2 allele(s) along with the transgene were then tracked throughout the production of a chicken ovalbumin-specific transgenic strain. The technique involved PCR-amplification of a DNA sequence encompassing the H-2 specific microsatellite followed by RFLP and heteroduplex analyses. This approach is likely to find wide application in the background checking of transgenic mice, especially in immunological research requiring a defined H-2 background.

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Transgene detection during early murine embryonic development after pronuclear microinjection

Cited by 16 publications

References 25 publications

Phage integrases for the construction and manipulation of transgenic mammals

Phage integrases for the construction and manipulation of transgenic mammals

Murine embryo development following cytoplasmic injection of linear and condensed DNA

TrackingH-2 alleles in transgenic mice by RFLP and heteroduplex analysis

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