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首页> 美国卫生研究院文献>Journal of Assisted Reproduction and Genetics >Transfection of the inner cell mass and lack of a unique DNA sequence affecting the uptake of exogenous DNA by sperm as shown by dideoxy sequencing analogues
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Transfection of the inner cell mass and lack of a unique DNA sequence affecting the uptake of exogenous DNA by sperm as shown by dideoxy sequencing analogues

机译：如双脱氧测序类似物所示内部细胞团的转染和缺乏影响精子摄取外源DNA的独特DNA序列的缺失

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>Purpose: The purpose of this study was to determine whether exogenous DNA internalized into blastocysts after transference from DNA-carrier sperm are localized at the inner cell mass or trophoblast cells and to identify differences in uptake of exogenous DNA fragments by sperm due to unique DNA sequences.>Methods: Mouse blastocysts at the hatching stage were exposed to migrating human sperm cells carrying exogenous DNA fragments synthesized from the E6–E7 conserved gene regions of human papillomavirus (HPV) types 16 and 18. After an interaction period of 2 hr, the transfected blastocysts were washed several times to remove extraneous sperm and the blastocysts were dissected into groups of cells derived from the inner cell mass and trophoblasts. The cells were analyzed by polymerase chain reaction (PCR) for the presence of HPV DNA fragments. In the second part of the experiment, thawed donor (N=10) sperm cells were pooled, washed, and divided into two fractions. The first (control) fraction was added with formalin and further divided and added with a35S-radiolabeled G, A, T, or C sequencing mixture. The second fraction was similarly treated but the formalin step was omitted from the treatment. After an hour of incubation at 37°C, the sperm specimens were washed several times by centrifugation and DNA extracted by the GeneReleaser method. The extracted DNA were processed on sequence gels, and the autoradiographs analyzed.>Results: Mouse blastocysts transfected by carrier sperm with DNA from HPV types 16 and 18 showed localization of the HPV DNA to both the inner cell mass and trophoblast cells. Negative controls consisting of untreated human sperm and untreated mouse blastocysts did not reveal any evidence of HPV DNA. The positive sperm control generated expected DNA fragments from HPV types 16 and 18. In the second experiment, the intensities of the DNA fragments in the G, A, T, and C columns from low to high molecular weights were not different from the positive control bands. Band intensities of the four sequencing columns were similar. Formalin pretreatment of the sperm inhibited uptake of the DNA fragments from the smallest to the largest DNA molecules.>Conclusions: Exogenous DNA taken into blastocysts are localized to both the inner cell mass and trophoblast cells. Only live sperm exhibited the capacity to carry various sizes of exogenous DNA, suggesting the involvement of active cell membrane mechanism in the transference process. The results showed that DNA fragments terminating in any of the four nucleotides were equally taken up by the sperm cell. Fragments of DNA produced by the sequencing reaction failed to identify a unique DNA sequence that would facilitate or inhibit the sperm from taking up exogenous DNA.
机译：>目的：本研究的目的是确定从DNA载体精子转移后内在化为胚泡的外源DNA是否位于内部细胞团或滋养层细胞中，并鉴定外源DNA片段摄取的差异>方法：孵化阶段的小鼠胚泡暴露于迁移的人类精子细胞中，这些细胞携带着从人类乳头瘤病毒（HPV）类型的E6–E7保守基因区域合成的外源DNA片段参见图16和18。在2小时的相互作用期后，将转染的胚泡洗涤数次以除去外来的精子，并将胚泡分成源自内部细胞团和滋养细胞的细胞群。通过聚合酶链反应（PCR）分析细胞中是否存在HPV DNA片段。在实验的第二部分中，将融化的供体（N = 10）精子细胞合并，洗涤并分成两部分。在第一个（对照）级分中加入福尔马林，进一步分开并添加 35 S放射性标记的G，A，T或C测序混合物。类似地处理第二部分，但是从处理中省略福尔马林步骤。在37°C下孵育一个小时后，通过离心将精子标本洗涤几次，并通过GeneReleaser方法提取DNA。提取的DNA在序列凝胶上进行处理，并进行放射自显影分析。>结果：用载体精子转染了HPV 16和18型DNA的小鼠胚泡显示HPV DNA既定位于内部细胞团又定位于滋养细胞。由未处理的人类精子和未处理的小鼠胚泡组成的阴性对照未显示出HPV DNA的任何证据。阳性精子对照从HPV 16和18型产生了预期的DNA片段。在第二个实验中，G，A，T和C柱中从低分子量到高分子量的DNA片段强度与阳性对照没有差异。乐队。四个测序柱的条带强度相似。福尔马林对精子的预处理抑制了从最小到最大的DNA分子对DNA片段的摄取。>结论：囊胚中吸收的外源DNA既定位于内部细胞团，又定位于滋养层细胞。只有活精子才具有携带各种大小的外源DNA的能力，这表明活性细胞膜机制参与了转移过程。结果显示，以四个核苷酸中的任何一个终止的DNA片段均被精子细胞吸收。测序反应产生的DNA片段未能鉴定出独特的DNA序列，该序列将促进或抑制精子吸收外源DNA。

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