410 USE OF SPERMATOZOA AS VECTORS OF EXOGENOUS DNA FOR <i>IN VITRO</i> PRODUCTION OF BOVINE TRANSGENIC EMBRYOS
Bibliographic record
Abstract
There are many methods to produce transgenic animals, although when considering bovine species, those methods offer low repeatability, high costs, and low transgene integration efficiency. To overcome these difficulties, sperm mediated gene transfer (SMGT) could be used as an alternative to produce transgenic embryos. This technology allows carrying exogenous DNA into the oocyte during the fertilization period, once spontaneous binding exists between spermatozoa and exogenous DNA. The aim of this study was to compare 4 methods for incorporating DNA into sperm cells—sperm incubation, capacitation, electroporation, and lipofection—to verify the efficiency of embryo production with exogenous DNA, employing spermatozoa as a vector. Cumulus–oocyte complexes (COCs) from abattoir-derived bovine ovaries were randomly divided into 5 groups (4 experimental groups: sperm incubation, sperm capacitation with calcium ionophore, electroporation, and lipofection; and 1 control group). In vitro maturation was performed in TCM-199 medium supplemented with 10% FCS, sodium pyruvate, gentamycin, FSH, hCG, and estradiol in an incubator at 39°C, in an atmosphere of 5% CO2 in air and high humidity for 24 h. After Percoll gradient (45/90%) separation at 600 g for 30 min, spermatozoa were washed in Talp Semen medium (200 g for 5 min). For in vitro fertilization (IVF), 5 × 106 spermatozoa were used to inseminate microdroplets with 20 matured oocytes from all groups: incubation with exogenous DNA for 1 h, sperm capacitation (250 nM of calcium ionophore) for 5 min followed by sperm incubation for 1 h, electroporation (500V), and lipofection (Effectene®; Qiagen, Mississauga, Ontario, Canada). The EYFP-Nuc plasmid (500 ng mL-1; Clontech, Mountain View, CA, USA) was used as exogenous DNA. Oocytes inseminated with non-treated sperm were considered as the control group. The presumptive zygotes were co-cultured with a granulosa cell monolayer in SOFaa medium in an incubator at 39°C, with an atmosphere of 5% CO2 in air and high humidity. The blastocyst rate was analyzed by ANOVA. Embryos from all experimental and control groups were subjected to PCR to detect internalized EYFP, using primers specific to this exogenous DNA, and considering positive embryos those that showed a fragment of 440 bp after electrophoresis. The fragment of 440 bp from positive embryos was sequenced to check correspondence to the EYFP. Electroporation (17.95%) and sperm capacitation (15.12%) were more efficient for EYFP-positive embryo production when compared to the lipofection protocol (6.25%). Sperm incubation (12.5%) did not show a significant difference from the other groups. Sequenced PCR-positive products for EYFP showed 100% homology with nucleotides of EYFP at GenBank. In conclusion, it is possible to use SMGT to deliver exogenous DNA into an oocyte at the time of IVF. This work was supported financially FAPESP 03/08542-5 and 03/07456-8 .
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".