Lactoferrin receptors in Gram-negative bacteria: an evolutionary perspective

In this short review, we outline the major events that led to the development of iron acquisition systems in Gram-negative bacteria and mammals since the beginning of life on earth. Naturally, the interaction between these organisms led to the development of a wonderfully complex set of protein systems used for competition over a once prevalent (but no longer) biocatalytic cofactor. These events led to the appearance of the lactoferrin gene, which has since been exploited into adopting countless new functions, including the provision of highly bactericidal degradation products. In parallel to lactoferrin’s evolution, evolving bacterial receptors have countered the bactericidal properties of this innate immunity protein.

Enhancing the antibacterial activities of sow milk via site-specific knock-in of a lactoferrin gene in pigs using CRISPR/Cas9 technology

Colostrum quality is a vital factor in mortality and growth performance for piglets. Lactoferrin is an immuno-active milk protein that contributes to the formation of a protective layer above intestinal mucosa, possesses the antibacterial and antiviral activities that are favorable for piglet development. However, there is a notable reduction in lactoferrin in sow milk during lactation after the first few days, which causes many piglets to fail to ingest enough colostrum thereby leading to an increase in piglet mortality. In this study, we successfully constructed genome-edited Large-White pigs with marker-free site-specific knock-in of lactoferrin gene in the 3′-end of Casein alpha-s1 via CRISPR/Cas9 mediated homologous recombination. Thus, the lactoferrin protein can be expressed in the mammary gland in the control of Casein alpha-s1 promoter. As expected, the lactoferrin protein in genetically modified pigs sustained high expression in both colostrum and milk when compared with wild-type pigs. Moreover, the bacterial plate assay indicated that the milk from genetically modified pigs showed bacteriostatic effects when compared with control pigs. Taken together, our study demonstrated that the milk from genetically modified pigs had antibacterial activity which may reduce the costs of veterinary drug and improve the surviving rate of piglets, which is promising for pig breeding.

Agrobacterium-mediated transformation of tobacco (Nicotiana tabacum L.) with human lactoferrin gene and analysis of transgenic lines

Aim. Obtaining of tobacco lines (Nicotiana tabacum L.) stably transformed with human lactoferrin gene (hLf), analysis of transgenic lines. Methods. Agrobacterium-mediated transformation of tobacco was carried out with the use of pBin35LF plasmid containing human lactoferrin gene under control of 35S promoter of cauliflower mosaic virus (CaMV 35S) and selective npt II gene encoding neomicyne phosphotransferase II providing the resistance to kanamycin. The selection of transgenic lines was carried out for 3 months on MS medium supplemented with 100 mg/l of kanamycin. Integration of lactoferrin gene was confirmed with the use of PCR with primers specific to hLf gene. The investigation of karyotype of transgenic lines was carried out after the staining of the root cells with 1 % solution of acetoorseine. Results. After selection, 2 transgenic tobacco lines with confirmed integration of hLf gene were obtained. The transformation efficiency was 6.4 %. The chromosome number in transgenic and control lines was 2n=48. Conclusions. The obtained transgenic tobacco lines have the similar morphology and karyotype to control lines, so they could be considered to use as plant systems for the expression of recombinant human lactoferrin. Keywords: human lactoferrin gene hLf, Nicotiana tabacum L., Agrobacterium-mediated transformation, PCR, chromosomes, transgenic plants.

Agrobacterium-mediated transformation of potato with human lactoferrin gene

Aim. Obtaining of the new Ukrainian potato cultivars with complex resistance to phytopathogens. Methods. Agrobacterium-mediated transformation of potato cultivars Vernisage and Zarevo with human lactoferrin gene possessing antimicrobial activity was carried out. For transformation, supervirulent A. tumefaciens strain EHA 105 with hLf gene and gene for the resistance to kanamycine was used. Selection was carried out for 3 months on MSK-S1 and MSK-S2 media supplemented with 100 mg/l kanamycine. Integration of lactoferrin gene in transgenic plant lines was confirmed by polymerase chain reaction with specific for hLf gene primers. Results. After selection, potato lines with the resistance to kanamycine were obtained. The transgenic potato lines with integrated into their genomes hLf gene were obtained. Integration of lactoferrin gene was confirmed for 1 line of Zarevo cultivar and 3 lines of Vernisage cultivar. Transformation frequency was of 6.8% for cv. Vernisage and 6.25% for cv. Zarevo. Conclusions. Selected potato lines would be used in further studies on their resistance to bacterial and fungal pathogens. Keywords: human lactoferrin gene hLf, Solanum tuberosum, Agrobacterium-mediated transformation, transgenic plants.