To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

Objective:To compare the predictive values of Bologna Outcome Algorithm for Stroke (BOAS) , Acute Stroke Registry and Analysis of Lausanne (ASTRAL) and Preadmission Comorbidities, Level of Consciousness, Age, and Focal Neurologic Deficit (PLAN) for stroke-associated pneumonia (SAP) and prognosis in elderly stroke patients in ICU.Methods:Using the convenient sampling method, a total of 198 elderly stroke patients who were hospitalized in ICU of Neurology Department of Henan Provincial People's Hospital were selected as the research objects from June 2017 to December 2019. According to whether SAP occurred within 4 weeks, patients were divided into SAP group, non-SAP group, severe SAP subgroup and mild SAP subgroup. According to the outcome within 4 weeks, they are divided into the poor prognosis group and the good prognosis group. The differences of BOAS, ASTRAL and PLAN scores among different groups were recorded and compared. ROC curve was used to analyze and compare the predictive performance of the three scales for SAP and its prognosis.Results:Pairwise positive correlation was found between BOAS, ASTRAL and PLAN scores ( P<0.05) . All the three scores of patients in SAP group were significantly higher than those in the non-SAP group, and ASTRAL and PLAN scores in severe SAP subgroup were higher than those in mild SAP subgroup, and the differences were significant ( P<0.05) . The area under ROC curve ( AUC) of BOAS, ASTRAL and PLAN scores were respectively 0.610, 0.692 and 0.705, and the AUC for predicting severe SAP were respectively 0.613, 0.661 and 0.709. In the SAP group and the non-SAP group, the scores of three scales of patients in the poor prognosis group were higher than those in the good prognosis group, and the differences were significant ( P<0.05) . The AUC of BOAS, ASTRAL and PLAN scores in the SAP group for predicting poor prognosis were respectively 0.736, 0.757 and 0.716, and AUC in the non-SAP group were respectively 0.699, 0.731 and 0.631. Conclusions:BOAS, ASTRAL and PLAN scores have certain predictive value for the SAP and prognosis of elderly patients in ICU. Among them, PLAN score has better predictive performance for SAP and ASTRAL has better predictive performance for prognosis.