Recombinase-mediated mouse transgenesis by intracytoplasmic sperm injection

Kaneko, Takehito; Moisyadi, Stefan; Suganuma, Ryota; Höhn, Barbara; Yanagimachi, Ryuzo; Pelczar, Pawel

doi:10.1016/j.theriogenology.2005.04.003

Cited by 29 publications

(35 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The difference from earlier successful reports of this method in the mouse [8], pig and goat [11] may be due to a different cytoplasmic environment in rat oocytes. At the local site where the sperm head was deposited, a lower concentration of magnesium ion and/or a higher concentration of ATP would dissociate the RecA from ssDNA/RecA, resulting in degradation of the ssDNA by cytoplasmic phosphodiesterases and nucleases.…”

contrasting

confidence: 62%

“…Maga et al [11] recently reported that recombinase-A protein (RecA)-coated ssDNA was more likely to be integrated into goat and pig genomes after pronuclear microinjection (6-and 10-fold higher, respectively). Very recently, Kaneko et al [8] have also reported that the ICSI-Tg using RecAcoated ssDNA produced transgenic mice at efficiencies of 2.9-7.7% of inseminated oocytes, while ICSI-Tg using the same ssDNA without RecA-coating resulted in no transgenic offspring. However, the mechanism by which the RecA-coating of exogenous DNA facilitates its integration into genomes remains unclear.…”

mentioning

confidence: 99%

“…There was no transgenic rat among 39 offspring that were born after transfer of 126 oocytes injected with ssEGFP/ RecA-bound sperm heads, while four transgenic rats were produced from transfer of 103 oocytes by the standard ICSI-Tg procedure. Kaneko et al [8] used 5 to 40 ng/µl EGFP DNA in the recombinase-mediated mouse transgenesis, with low cleavage rates (26 to 36%) but high offspring rates (28-67%). The final concentration of EGFP DNA in rat ICSI-Tg (0.1 ng/µl) was determined based on our previous study using the same DNA construct [7].…”

mentioning

confidence: 99%

See 2 more Smart Citations

No Effect of Recombinase-Mediated DNA Transfer on Production Efficiency of Transgenic Rats

et al. 2006

View full text Add to dashboard Cite

It was reported that recombinase-Although pronuclear DNA microinjection is the most convenient approach to produce transgenic animals, the low integration of exogenous DNA into genomes is still an obstacle to the widening of the transgenic technology. In general, the proportion of microinjected zygotes developing into transgenic offspring is <5% in rodents [1,5], and <1% in large domestic species [17,22]. Another approach to production of transgenic animals is intracytoplasmic sperm injection (ICSI)-mediated transgenesis (ICSI-Tg) [16], in which <7% of inseminated oocytes develop into transgenic mice [16,20] and rats [7,9]. Maga et al. [11] recently reported that recombinase-A protein (RecA)-coated

show abstract

contrasting

confidence: 62%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

No Effect of Recombinase-Mediated DNA Transfer on Production Efficiency of Transgenic Rats

et al. 2006

View full text Add to dashboard Cite

show abstract

“…In this method, recombinases or transposases are injected into mouse oocytes to increase the efficiency of transgene integration into the genome. We have demonstrated that the bacterial recombinase protein RecA (Kaneko et al 2005) and a mutated hyperactive Tn5 transposase protein (*Tn5p) ) both increase transgenesis several fold as compared to conventional methods such as pronuclear microinjection (Nakanishi et al 2002) and traditional ICSI-Tr (Perry et al 1999). …”

Section: Intracytoplasmic Sperm Injection-mediated Transgenesismentioning

confidence: 99%

“…Among approaches utilizing protein recombinases (RecA) (Kaneko et al 2005) or transposases, the hyperactive Tn5 transposase protein (*Tn5p) was by far the most efficient method for introducing the tg in a transposon along with spermatozoa into unfertilized oocytes (TN:ICSI) ). In our hands, this approach dramatically increased the efficiency of producing transgenic mice, with 11% of eggs injected and 22% of live births resulting in transmission of the tg DNA and over 75% of transgenic mice expressing the EGFP tg.…”

Section: Active Transgenesismentioning

confidence: 99%

Active integration: new strategies for transgenesis

Shinohara

Kaminski²,

Segal

et al. 2007

Transgenic Res

Self Cite

View full text Add to dashboard Cite

This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed "Active Transgenesis" to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac's specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.

show abstract

Generation of Transgenic Rats by Ooplasmic Injection of Sperm Cells Exposed to Exogenous DNA

Hirabayashi

Hochi

2009

Methods in Molecular Biology

View full text Add to dashboard Cite

Intracytoplasmic sperm injection (ICSI) has been successfully achieved in mice and rats using a piezo-driven injection pipette, with the offspring rate of >30%. The ICSI technique was applied not only to rescue infertile male strains but also to produce transgenic rodents. The ICSI-mediated DNA transfer, that the sperm heads and exogenous DNA solution are mixed and co-injected into ooplasm, has been equally effective to the conventional pronuclear DNA microinjection. Production efficiency of transgenic founders by the ICSI-mediated DNA transfer was comparable between mice and rats, while the optimal DNA concentration was lower in rats than mice. The production efficiency was improved when membrane structure of sperm heads was partially disrupted by detergent or ultrasonic treatment before exposure to the exogenous DNA solution. Exogenous DNAs with various chain lengths were stably integrated into the rodent genomes of various genetic backgrounds by this method. The ICSI-mediated DNA transfer in which the preparation of pronuclear-stage fertilized zygotes is not required would be alternative to conventional pronuclear DNA microinjection.

show abstract

Recombinase-mediated mouse transgenesis by intracytoplasmic sperm injection

Cited by 29 publications

References 25 publications

No Effect of Recombinase-Mediated DNA Transfer on Production Efficiency of Transgenic Rats

No Effect of Recombinase-Mediated DNA Transfer on Production Efficiency of Transgenic Rats

Active integration: new strategies for transgenesis

Generation of Transgenic Rats by Ooplasmic Injection of Sperm Cells Exposed to Exogenous DNA

Contact Info

Product

Resources

About