Cattle with a precise, zygote-mediated deletion safely eliminate the major milk allergen beta-lactoglobulin.

We applied precise  zygote-mediated genome editing to eliminate beta-lactoglobulin (BLG), a major allergen in cows' milk. To efficiently generate LGB knockout cows, biopsied embryos were screened to transfer only appropriately modified embryos. Transfer of 13 pre-selected embryos into surrogate cows resulted in the birth of three calves, one dying shortly after birth. Deep sequencing results confirmed conversion of the genotype from wild type to the edited nine bp deletion by more than 97% in the two male calves. The third calf, a healthy female, had in addition to the expected nine bp deletion (81%), alleles with an in frame 21 bp deletion (<17%) at the target site. While her milk was free of any mature BLG, we detected low levels of a BLG variant derived from the minor deletion allele. This confirmed that the nine bp deletion genotype completely knocks out production of BLG. In addition, we showed that the LGB knockout animals are free of any TALEN-mediated off-target mutations or vector integration events using an unbiased whole genome analysis. Our study demonstrates the feasibility of generating precisely biallelically edited cattle by zygote-mediated editing for the safe production of hypoallergenic milk.

Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9.

Goat's milk, considered a substitute for cow's milk, has a high nutritional value. However, goat's milk contains various allergens, predominantly ß-lactoglobulin (BLG). In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs) into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%-9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/µL sg1) and 0% (10 ng/µL sg2) and efficiencies of dual sgRNAs were 25.0% (25 ng/µL sg2+sg3 group) and 28.6% (50 ng/µL sg2+sg3 group). Relative expression of BLG in BLG knock-out goat mammary glands significantly (p < 0.01) decreased as well as other milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p < 0.05). As expected, BLG protein had been abolished in the milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4), and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research.

Gene targeting by TALEN-induced homologous recombination in goats directs production of ß-lactoglobulin-free, high-human lactoferrin milk.

ß-Lactoglobulin (BLG) is a major goat's milk allergen that is absent in human milk. Engineered endonucleases, including transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases, enable targeted genetic modification in livestock. In this study, TALEN-mediated gene knockout followed by gene knock-in were used to generate BLG knockout goats as mammary gland bioreactors for large-scale production of human lactoferrin (hLF). We introduced precise genetic modifications in the goat genome at frequencies of approximately 13.6% and 6.09% for the first and second sequential targeting, respectively, by using targeting vectors that underwent TALEN-induced homologous recombination (HR). Analysis of milk from the cloned goats revealed large-scale hLF expression or/and decreased BLG levels in milk from heterozygous goats as well as the absence of BLG in milk from homozygous goats. Furthermore, the TALEN-mediated targeting events in somatic cells can be transmitted through the germline after SCNT. Our result suggests that gene targeting via TALEN-induced HR may expedite the production of genetically engineered livestock for agriculture and biomedicine.

Efficient gene knockout in goats using CRISPR/Cas9 system.

The CRISPR/Cas9 system has been adapted as an efficient genome editing tool in laboratory animals such as mice, rats, zebrafish and pigs. Here, we report that CRISPR/Cas9 mediated approach can efficiently induce monoallelic and biallelic gene knockout in goat primary fibroblasts. Four genes were disrupted simultaneously in goat fibroblasts by CRISPR/Cas9-mediated genome editing. The single-gene knockout fibroblasts were successfully used for somatic cell nuclear transfer (SCNT) and resulted in live-born goats harboring biallelic mutations. The CRISPR/Cas9 system represents a highly effective and facile platform for targeted editing of large animal genomes, which can be broadly applied to both biomedical and agricultural applications.

Anomalous neutrophil granule distribution in a patient with lactoferrin deficiency: pertinence to the respiratory burst.

Neutrophils from a patient with lactoferrin deficiency were examined and the quantity and subcellular localization of protein markers were determined on Percoll density gradients. Distribution of azurophilic and specific granule markers was abnormal in that azurophilic granules were lighter than normal and appeared in the fraction of the gradient where normally the specific granules sediment. The specific granule membrane markers, cytochrome b-235 and its associated flavoprotein, were abnormally distributed in the gamma fraction, the site of the plasma membrane marker alkaline phosphatase. Thus, the b-cytochrome-flavoprotein complex had either been incorporated into the plasma membrane or was still present in the membranes of granules that were abnormally light and cosedimented with the plasma membranes. This is of particular interest in regard to the patient's respiratory burst oxidase function, since the b-cytochrome/flavoprotein complex normally translocates from the specific granules to the plasma membrane to constitute the active respiratory burst oxidase. The functional consequences of this abnormal distribution are discussed, as is the importance of characterizing both intragranular enzymatic markers and granule membrane proteins to define granular disorders.

Lactoferrin-deficient neutrophil polymorphonuclear leucocytes in leukaemias: a semiquantitative and ultrastructural cytochemical study.

Semiquantitative analysis of lactoferrin deficiency in neutrophil polymorphonuclear leucocytes in various haematological and non-haematological disease was carried out by scoring polymorphonuclear leucocytes stained for lactoferrin by the immunoperoxidase method. The staining patterns for lactoferrin were classified into four types (0-III) based on the intensity of reaction, and the sum of the ratings of 100 polymorphonuclear leucocytes was considered as "lactoferrin score" with a possible range of 0-300. As a result, significantly low lactoferrin-scores were frequently observed in acute leukaemias and the acute phase of chronic leukaemias. Of 35 cases with leukaemias, lactoferrin-negative polymorphonuclear leucocytes (type 0) were observed in the following cases: eight cases of acute myelogenous leukaemia (8/14), a case of chronic myelogenous leukaemia (1/10) in blast crisis, one of acute promyelocytic leukaemia (1/1), one of acute monocytic leukaemia (1/2), and a case of chronic myelomonocytic leukaemia (1/2) in a transitional phase to an acute myelomonocytic leukaemia. In two cases of acute myelogenous leukaemia, in which the majority of polymorphonuclear leucocytes were negative for lactoferrin, ultrastructural cytochemical study revealed total lack of specific granules in these polymorphonuclear leucocytes. This suggests that lactoferrin is localised in the specific granules of neutrophils as has been postulated previously by others.

Abnormal distribution of complex carbohydrates in neutrophils of a patient with lactoferrin deficiency.

Previous studies have identified patients with susceptibility to bacterial infection associated with lactoferrin deficiency in dysmorphic neutrophils containing abnormal or no secondary granules and abnormal nuclear segmentation. We have investigated the subcellular distribution of vicinal glycol-containing complex carbohydrates in marrow and blood myeloid cells of such a patient using the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining method and have examined the response of these neutrophils to the degranulating agents N-formylmethionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA). As in normal specimens, immature primary granules were strongly PA-TCH-SP reactive; however, unlike normal specimens, masking of PA-TCH-SP reactivity did not occur in mature primary granules. Endoplasmic reticulum demonstrated moderately strong PA-TCH-SP staining, in contrast to absent staining of this organelle in normal promyelocytes and consistent with abnormal primary granule genesis. Small abnormal elongated granules (0.1-0.2 micron in diameter) were identified at the myelocyte state of development and were the predominant granule type in late neutrophils. These granules were identified as secondary granules on the basis of their PA-TCH-SP positivity and were differentiated from primary and tertiary granules on the basis of a lack of peroxidase, acid phosphatase, and sulfate staining. When the neutrophils were exposed to PMA, cell aggregation occurred, and the abnormal granules degranulated in a manner similar to the degranulation observed with normal secondary granules. Although PA-TCH-SP staining of the plasma membrane appeared normal, a decrease in FMLP receptors was demonstrated. Thus, a defect(s) is present in complex carbohydrate distribution and staining that involves primary and secondary granules and possibly the plasmalemma of neutrophils from this patient. This results in abnormal packaging of primary granules and synthesis of normal numbers of secondary granules that are qualitatively and morphologically abnormal, but can be recruited to degranulate with PMA.

Lactoferrin deficiency as a consequence of a lack of specific granules in neutrophils from a patient with recurrent infections. Detection by immunoperoxidase staining for lactoferrin and cytochemical electron microscopy.

Neutrophils from a boy suffering from recurrent infections were found to be totally deficient in specific granules when studied by electron microscopy. In contrast, myeloperoxidase-containing azurophil granules were increased in number. This deficiency of specific granules could be detected at the light-microscopic level using an immunocytochemical technique to demonstrate the absence of lactoferrin. Neutrophils also exhibited abnormal nuclear segmentation, nuclear clefts, an abnormally weak cytochemical reaction for alkaline phosphatase, and an increased number of mitochondria and ribosomes. Some granulocytic precursors were abnormal, and many of these cells were phagocytosed by macrophages in the bone marrow. Despite these multiple abnormalities and the history of severe pyogenic infection, the in vitro bactericidal capacity of the neutrophils was within normal limits, and normal degranulation of azurophil granules occurred following phagocytosis. The precise mechanism by which the deficiency of specific granules in this patient led to an enhanced in vivo susceptibility to infection therefore remains obscure. However, attention is drawn to the fact that in three previously described cases of specific granule deficiency a history of recurrent infections was present.