Transgene detection during early murine embryonic development after pronuclear microinjection.
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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R L
RL
Department of Chemical Engineering, Virginia Polytechnic Institute, Blacksburg.
Canseco
R S
RS
Russell
C G
CG
Johnson
J L
JL
Velander
W H
WH
Gwazdauskas
F C
FC
D000818:Animals; D046508:Culture Techniques; D004247:DNA / Q000235:genetics; D005314:Embryonic and Fetal Development / Q000235:genetics*; D005260:Female; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D008894:Milk Proteins / Q000235:genetics*; D053652:Nuclear Transfer Techniques; D016133:Polymerase Chain Reaction
D008894:Milk Proteins; C024471:whey acidic proteins; D004247:DNA