Pretreatment of murine embryos prior to the polymerase chain reaction

Fuller, D.T.; Kochevar, Deborah T.; Brumbaugh, Gordon W.; Womack, James E.; Kraemer, D.C.

doi:10.1016/0093-691x(93)90076-h

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“…The band intensity with the ethanol and detergentprotease lysis suggests a more efficient amplification of the embryonic DNA. Our results appear to be more effective for visualization of DNA products than those of Fuller et al (1993), who used only heat and Proteinase K pretreatment of one-cell murine zygotes. The detection of PCR products from WAP gene amplification in later stage embryos was greater than that of earlier ova, suggesting a copy-number-dependent amplification efficiency.…”

Section: Discussionmentioning

confidence: 47%

Transgene detection during early murine embryonic development after pronuclear microinjection

Page

Canseco

Russell

et al. 1995

Transgenic Research

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The polymerase chain reaction (PCR) technique was used to detect a whey acidic protein (WAP) gene and transgene presence in mouse ova cultured to various stages of development after pronuclear microinjection at the one-cell stage. The PCR technique detected an endogenous 442 bp WAP DNA sequence in 78% of one-cell, 88% of two-cell and 94% of four-cell ova, and in 95% of morulae and 97% of blastocysts. The heterologous WAP-human protein C transgene was detected in 88% of one-cell, 88% of two-cell and 44% of four-cell ova, and in 40% of morulae and 29% of blastocysts. For comparison, the integration frequency for transgenic mouse production using the same DNA construct was 22%. After five days of in vitro culture, embryos that were either developmentally arrested or fragmented were tested for the presence of the transgene. The injected construct was detected in 83% of arrested one-cell, 85% of arrested two-cell, and 85% of fragmented ova. In culture, only 28% of zygotes microinjected with DNA developed to the blastocysts stage compared to 74% of noninjected zygotes, while 63% of zygotes developed to the blastocyst stage after injection of buffer alone. Pronuclear injection of the transgene at concentrations of 1.5, 15 and 50 micrograms ml-1 resulted in 28, 11 and 9% development to blastocysts and 29, 86 and 88% transgene detection, respectively. Transgene detection was 85, 96 and 97% in degenerate embryos at the respective doses of DNA. These data show that pronuclear microinjection of the transgene is detrimental to subsequent embryonic development.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 47%