31 CONSTRUCTION OF A TARGETING VECTOR SPECIFIC FOR THE BOVINE BETA-CASEIN GENE
Bibliographic record
Abstract
Although using livestock as animal bioreactors is a powerful tool to produce valuable therapeutic proteins in the milk, there are still some limitations to the technology such as a low frequency of transgenesis and a low expression of the transgenes by random integration. In this study, we constructed gene-targeting vectors for a mammary gland-specific gene and then obtained two homologous recombinant cell clones after transfection of the vectors into bovine somatic cells. Two targeting vector cassettes, BCKI I and BCKI II, which have homology regions for a bovine beta-casein gene, were constructed. The beta-casein gene is expressed only in the mammary gland and is the most abundant milk protein in the cow. The targeting sequence lengths of the BCKI I and BCKI II vector cassettes were 13.1 kb and 9.1 kb, respectively, and contained different long arm lengths. A neo gene was inserted into the vectors as a selection marker, and a few restriction enzyme sites were made in front of the neo gene. The human thrombopoietin (TPO) gene was inserted into the restriction enzyme sites of the vector cassettes, named BCTPOKI I and BCTPOKI II vectors. The BCTPOKI I and BCTPOKI II vectors were transfected into bovine embryonic fibroblasts (bEF) and ear skin fibroblasts (bESF) using Lipofectamine™ 2000 reagent (Invitrogen, Seoul, South Korea). In order to determine the highest transfection efficiency, a variety of factors such as DNA concentration, lipid volume, and exposure time to DNA-liposome complexes based on the manufacturers' guideline, was optimized. The 2:1 and 1:2 ratios of DNA (µg) to transfection reagent (µL) were efficient for bEF and bESF, respectively, under overnight exposure to DNA-Lipofectamine™ 2000 reagent. Seventeen percent (51/304) of bESF clones and 6% (9/149) of bEF clones were normally expanded into passage 8. PCR and Southern blotting indicated that 6.3% (2/32) of the clones carrying with BCTPOKI II vectors was homologously targeted at the beta-casein gene. However, none (0/60) of the clones carrying BCTPOKI I was targeted. Additionally, both of the targeted clones were from bESF. When the targeted cells were transferred into enucleated oocytes and cultured, 83% (43/52) of the cloned embryos were transgenic. Thus, we found that homologous recombinant events using gene-targeting vectors might be dependent on cell types, vector sizes, and transfection procedures. In conclusion, mammary gland-specific gene-targeting vectors coupled with somatic cell nuclear transfer technology will be very useful for developing animal bioreactors that produce therapeutic proteins in milk.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| 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 | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
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 teacher head, 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".