Generation of cattle knockout for galactose-α1,3-galactose and N-glycolylneuraminic acid antigens.

Two well-characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine-derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty-three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double-KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double-KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet.

Monoclonal antibody specific to alpha-2-->3-linked deaminated neuraminyl beta-galactosyl sequence.

Fusion of spleen cells from a BALB/c mouse immunized with KDN alpha 2-->3Gal beta 1-->4Glc beta 1-->1Cer ((KDN)GM3) with P3-X63 Ag8.U1 (P3U1) mouse myeloma cells yielded a hybrid cell line that produced monoclonal antibody that bound to (KDN)GM3, but not to Neu5Ac alpha 2-->3Gal beta 1-->4Glc-beta 1-->1Cer ((Neu5Ac)GM3). The specificity of the monoclonal antibody was determined chiefly by the enzyme-linked immunosorbent assay procedure. This antibody was found to react most strongly with (KDN)GM3 and less strongly with a glycoprotein containing a number of KDN alpha 2-->3Gal beta 1-->3-GalNAc alpha 1-->3[8KDN alpha 2-->)n-->6]GalNAc alpha 1-->chains (< n > av = approximately 3). The results indicated that the monoclonal antibody (designated mAb.kdn3G) specifically and effectively recognized a disaccharide structure, KDN alpha 2-->3Gal beta 1-->, and specifically discriminated (KDN)GM3 from (Neu5Ac)GM3. The mAb.kdn3G was used to localize (KDN)GM3 in rainbow trout sperm by the indirect immunofluorescence procedure and the antigen was shown to be mostly, if not completely, associated with the external surface of the entire plasma membrane of rainbow trout sperm. The potential utility of mAb.kdn3G is addressed in searching for KDN-glycoconjugates which contain glycan units having the KDN alpha 2-->3Gal beta 1-->epitope structure.