CD18 Gene Research Articles

Association of integrin-β2 polymorphism and expression with the risk of rheumatoid arthritis and osteoarthritis in Egyptian patients

BackgroundThe genetic architecture of rheumatoid arthritis (RA) and osteoarthritis (OA) are still unclear. Although RA and OA have quite different causes, they share synovial inflammation, risk factors, and some disease-associated genes, including the integrin subunit β2 (ITGB2)/CD18 gene involved in extracellular matrix interactions and immune cell signaling. However, the functional role of ITGB2 genetic variants, its circulating expression pattern, and their clinical usefulness in RA and OA remain unexplored. Our study appraised the association of ITGB2 rs2070946 single nucleotide polymorphism with the vulnerability to RA and OA and its influence on ITGB2 mRNA expression, along with the potential of serum ITGB2 expression in RA and OA diagnosis.MethodsThis study included 70 RA patients, 70 primary OA patients, and 60 healthy volunteers. Genotyping and gene expression analysis were performed using qPCR. Bioinformatics analysis was employed to construct the protein-protein interaction (PPI) network of ITGB2.ResultsSerum ITGB2 mRNA expression was upregulated in both RA and OA compared to healthy controls. ITGB2 rs2070946 was associated with escalating risk of both diseases. RA patients harboring the rs2070946 CC or TC + CC genotypes had higher serum ITGB2 expression than the TT genotype carriers. Likewise, OA patients having the minor homozygote CC genotype had higher serum ITGB2 expression than those carrying the TT, TC or TT + TC genotypes. Serum ITGB2 expression showed profound diagnostic potential for RA and OA in receiver-operating characteristic analysis. In RA, serum ITGB2 expression positively correlated with rheumatoid factor and disease activity score 28 (DAS28). The ITGB2-PPI network enriched in cell-cell adhesion, ICAM-3 receptor activity, T-cell activation, leukocyte adhesion, complement binding, and NF-κB, tumor necrosis factor, and interleukin signaling pathways.ConclusionThese findings embrace the impact of ITGB2 rs2070946 as a novel genetic biomarker of both RA and OA, which could alter the ITGB2 expression. Serum ITGB2 expression could aid in timely diagnosis of RA and OA.

Heterotropic roles of divalent cations in the establishment of allostery and affinity maturation of integrin αXβ2.

SUMMARYAllosteric activation and silencing of leukocyte β2-integrins transpire through cation-dependent structural changes, which mediate integrin biosynthesis and recycling, and are essential to designing leukocyte-specific drugs. Stepwise addition of Mg2+ reveals two mutually coupled events for the αXβ2 ligand-binding domain—the αX I-domain—corresponding to allostery establishment and affinity maturation. Electrostatic alterations in the Mg2+-binding site establish long-range couplings, leading to both pH− and Mg2+-occupancy-dependent biphasic stability change in the αX I-domain fold. The ligand-binding sensorgrams show composite affinity events for the αX I-domain accounting for the multiplicity of the αX I-domain conformational states existing in the solution. On cell surfaces, increasing Mg2+ concentration enhanced adhesiveness of αXβ2. This work highlights how intrinsically flexible pH− and cation-sensitive architecture endows a unique dynamic continuum to the αI-domain structure on the intact integrin, thereby revealing the importance of allostery establishment and affinity maturation in both extracellular and intracellular integrin events.

β2 Integrins on Dendritic Cells Modulate Cytokine Signaling and Inflammation-Associated Gene Expression, and Are Required for Induction of Autoimmune Encephalomyelitis.

Heterodimeric β2 integrin surface receptors (CD11a-d/CD18) are specifically expressed by leukocytes that contribute to pathogen uptake, cell migration, immunological synapse formation and cell signaling. In humans, the loss of CD18 expression results in leukocyte adhesion deficiency syndrome (LAD-)1, largely characterized by recurrent severe infections. All available mouse models display the constitutive and ubiquitous knockout of either α or the common β2 (CD18) subunit, which hampers the analysis of the cell type-specific role of β2 integrins in vivo. To overcome this limitation, we generated a CD18 gene floxed mouse strain. Offspring generated from crossing with CD11c-Cre mice displayed the efficient knockdown of β2 integrins, specifically in dendritic cells (DCs). Stimulated β2-integrin-deficient splenic DCs showed enhanced cytokine production and the concomitantly elevated activity of signal transducers and activators of transcription (STAT) 1, 3 and 5, as well as the impaired expression of suppressor of cytokine signaling (SOCS) 2–6 as assessed in bone marrow-derived (BM) DCs. Paradoxically, these BMDCs also showed the attenuated expression of genes involved in inflammatory signaling. In line, in experimental autoimmune encephalomyelitis mice with a conditional DC-specific β2 integrin knockdown presented with a delayed onset and milder course of disease, associated with lower frequencies of T helper cell populations (Th)1/Th17 in the inflamed spinal cord. Altogether, our mouse model may prove to be a valuable tool to study the leukocyte-specific functions of β2 integrins in vivo.

Exploration of Polymorphism Analysis of CD18 Gene with PCR-RFLP Technique in Friesian Holstein Crossbreed Dairy Cows in Batu, East Java, Indonesia

Exploration of Polymorphism Analysis of CD18 Gene with PCR-RFLP Technique in Friesian Holstein Crossbreed Dairy Cows in Batu, East Java, Indonesia

BLAD and CVM in the genetic structure of the cattle breeding stock in the Sverdlovsk region

The identification of lethal and unwanted mutations in populations of black-and-white cattle breeding remains relevant, despite the fact that, in modern conditions, breeding bulls are being investigated for a wide range of known genetic anomalies. The broodstock not only of commercial farms, but also of breeding ones, remains not examined for mutations that are associated with economic losses due to the nonviability of the offspring and perinatal losses, the causes of which cannot be identified excluding infectious and alimentary factors. The genotyping of the broodstock carried out for the first time in the Sverdlovsk region confirms the presence in herds of mutant forms of the CD18 and SLC35A3 gene alleles in a latent state. In the studied herds, regardless of their breeding status, the carriage of BLAD and CVM anomalies is observed from 2.08% to 13.51% and from 2.70% to 7.55%, respectively. In some farms, carriage of two mutations reaches 16.21%. The results obtained demonstrate the prevalence of carriage of the studied alleles in most genealogical lines of bulls of the Holstein root. Thus, in the Vis Bek Aydial 933122 line, six daughters from six sire bulls were found to carry these mutant loci, in the Reflection Sovering 198998 lines in eight daughters from three sire bulls and in the Montvik Chief 95679 line in three daughters from three bulls. Lethal alleles of both genes were found in each line. In breeding organizations, it is necessary to conduct research on the carriage of lethal and unwanted genes, especially in a group of cows-mothers of replacement bulls, to prevent the spread of anomalies in the breed.

Detection of the CD18 gene mutation as a marker of BLAD genetic disorder of Holstein-Friesian cattle in West Java

<p class="MDPI17abstract"><strong>Objective: </strong>Bovine Leukocyte Adhesion Deficiency (BLAD) is a genetic disorder in Holstein-Friesian (HF) cattle that have a significant economic impact on dairy cattle breeding. Mutation c.383A>G in Cluster of Differentiation Molecule 18 (CD18) gene was known as related to BLAD. This study aimed to detect the CD18 gene mutation that causes BLAD of HF cattle in the West Java Province.</p><p class="MDPI17abstract"><strong>Methods: </strong>The genomic DNA was extracted from 88 blood samples of HF cattle from Cibungbulang-Bogor (n=34), Ciampea-Bogor (n=31), and Sukabumi (n=23) by the GB300 DNA extraction kit, Geneaid<sup>TM</sup>. The CD18 gene mutation was detected by PCR-RFLP analysis using the <em>Taq</em>I restriction endonuclease.<strong></strong></p><p class="MDPI17abstract"><strong>Results: </strong>All samples in this study produced two fragments DNA, i.e., 359 bp and 260 bp with the monomorphic homozygote genotype (BB). HF cattle with BB genotype indicated as normal cattle and did not carry BLAD.<strong></strong></p><strong>Conclusions: </strong>All samples in the three populations of West Java were free of BLAD genetic disorder. Identification of BLAD on HF cattle in other places, including bulls in the Center of Semen Production in Indonesia was needed to prevent this genetic disorder.

MSCs rescue impaired wound healing in a murine LAD1 model by adaptive responses to low TGF‐β1 levels

Mutations in the CD18 gene encoding the common β‐chain of β2 integrins result in impaired wound healing in humans and mice suffering from leukocyte adhesion deficiency syndrome type 1 (LAD1). Transplantation of adipose tissue‐derived mesenchymal stem cells (MSCs) restores normal healing of CD18−/− wounds by restoring the decreased TGF‐β1 concentrations. TGF‐β1 released from MSCs leads to enhanced myofibroblast differentiation, wound contraction, and vessel formation. We uncover that MSCs are equipped with a sensing mechanism for TGF‐β1 concentrations at wound sites. Low TGF‐β1 concentrations as occurring in CD18−/− wounds induce TGF‐β1 release from MSCs, whereas high TGF‐β1 concentrations suppress TGF‐β1 production. This regulation depends on TGF‐β receptor sensing and is relayed to microRNA‐21 (miR‐21), which subsequently suppresses the translation of Smad7, the negative regulator of TGF‐β1 signaling. Inactivation of TGF‐β receptor, or overexpression or silencing of miR‐21 or Smad7, abrogates TGF‐β1 sensing, and thus prevents the adaptive MSC responses required for tissue repair.

β2 Integrins-Multi-Functional Leukocyte Receptors in Health and Disease.

β2 integrins are heterodimeric surface receptors composed of a variable α (CD11a-CD11d) and a constant β (CD18) subunit and are specifically expressed by leukocytes. The α subunit defines the individual functional properties of the corresponding β2 integrin, but all β2 integrins show functional overlap. They mediate adhesion to other cells and to components of the extracellular matrix (ECM), orchestrate uptake of extracellular material like complement-opsonized pathogens, control cytoskeletal organization, and modulate cell signaling. This review aims to delineate the tremendous role of β2 integrins for immune functions as exemplified by the phenotype of LAD-I (leukocyte adhesion deficiency 1) patients that suffer from strong recurrent infections. These immune defects have been largely attributed to impaired migratory and phagocytic properties of polymorphonuclear granulocytes. The molecular base for this inherited disease is a functional impairment of β2 integrins due to mutations within the CD18 gene. LAD-I patients are also predisposed for autoimmune diseases. In agreement, polymorphisms within the CD11b gene have been associated with autoimmunity. Consequently, β2 integrins have received growing interest as targets in the treatment of autoimmune diseases. Moreover, β2 integrin activity on leukocytes has been implicated in tumor development.

Screening of Hardhenu Crossbred, Sahiwal and Hariana Bulls for Autosomal Recessive Genetic Disorder Bovine Leukocyte Adhesion Deficiency (BLAD)

The present study involved screening of Hardhenu crossbred, Sahiwal and Hariana bulls for autosomal recessive genetic disorder i.e Bovine Leukocyte Adhesion Deficiency (BLAD) using PCR based techniques. BLAD is a lethal autosomal recessive disease caused due to a point mutation (A→G) at nucleotide 383 in the CD18 gene which results in substitution of aspartic acid with glycine in the adhesion glycoprotein CD18. All animals under study were maintained at Cattle Breeding Farm, LalaLajpat Rai University of Veterinary and Animal Science (LUVAS), Hisar, India. About 5 ml blood was collected aseptically from jugular vein puncture into EDTA containing tubes, transported to the animal genomics laboratory and stored at-20°C until genomic DNA extraction, which was carried out using Phenol Chloroform method. The targeted genomic area of interest was amplified by using simple PCR technique. The amplified PCR products were digested with TaqI restriction enzyme. The targeted amplicon of 357 bp after digestion of the PCR product, the normal allele in unaffected bull produced two fragments of 201 bp and 156 bp. All the animals under study were found to be free from Bovine Leukocyte Adhesion Deficiency. The molecular screening revealed that none of the bulls screened under study was carrier for BLAD. The present study suggests that the PCR and PCR-RFLP based technique could be employed as an efficient technique for screening of various genetic disorders for identification of carriers or affected animals before using them in breeding programs to minimize the spread of defective allele.

Efficiency of different fragment lengths of the ubiquitous chromatin opening element HNRPA2B1-CBX3 in driving human CD18 gene expression within self-inactivating lentiviral vectors for gene therapy applications

Patients with leukocyte adhesion deficiency type 1 (LAD1) suffer from life-threatening bacterial infections due to mutations in the common β2 integrin subunit (CD18/ITGB2 gene).We tested different fragments of the ubiquitous chromatin opening element (UCOE) from the human HNRPA2B1-CBX3 locus for their efficiency in driving the human CD18 gene expression and compared it with that of an elongation factor 1 alpha promoter (EF1αL, 1169 bp; EF1αS 248 bp) and a murine stem cell virus (MSCV) promoter within the context of the same lentiviral vector backbone. These vectors were tested in vitro for the human CD18 gene expression on the surface of CD34+ hematopoietic stem cells (HSCs) isolated from both moderate and severe LAD1 patients. Among the promoters tested in the patients' CD34+ HSCs, only U631 bp, U652 bp, U1262 bp, 5′ 2.2 kb A2UCOE and EF1αS resulted in higher percentage of CD18+CD34+ cells comparable to that of the MSCV promoter. The U655 bp, U723 bp, U1296 bp, U2598 bp and EF1αL promoters resulted in comparatively lower numbers of CD18+CD34+ cells. This study would be useful in investigating the human CD18 gene expression in an ex vivo experiment to demonstrate the phenotypic correction of LAD1 in a pre-clinical model.

Macrophage β2-Integrins Regulate IL-22 by ILC3s and Protect from Lethal Citrobacter rodentium-Induced Colitis

SUMMARYβ2-integrins promote neutrophil recruitment to infected tissues and are crucial for host defense. Neutrophil recruitment is defective in leukocyte adhesion deficiency type-1 (LAD1), a condition caused by mutations in the CD18 (β2-integrin) gene. Using a model of Citrobacter rodentium (CR)-induced colitis, we show that CD18−/− mice display increased intestinal damage and systemic bacterial burden, compared to littermate controls, ultimately succumbing to infection. This phenotype is not attributed to defective neutrophil recruitment, as it is shared by CXCR2−/− mice that survive CR infection. CR-infected CD18−/− mice feature prominent upregulation of IL-17 and downregulation of IL-22. Exogenous IL-22 administration, but not endogenous IL-17 neutralization, protects CD18−/− mice from lethal colitis. β2-integrin expression on macrophages is mechanistically linked to Rac1/ROS-mediated induction of noncanonical-NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome-dependent IL-1β production, which promotes ILC3-derived IL-22. Therefore, β2-integrins are required for protective IL-1β-dependent IL-22 responses in colitis, and the identified mechanism may underlie the association of human LAD1 with colitis.

Study of Genetic Polymorphism of Cd18 Gene in Tunisian Holstein Cows

Study of Genetic Polymorphism of Cd18 Gene in Tunisian Holstein Cows

Identification of heterozygous cases of Bovine leukocyte adhesion deficiency (BLAD) in Indian Holstein crossbred bulls

BLAD is an Autosomal genetic disorder mainly in Holstein and its crosses are major concern worldwide as heterozygous (carrier) looks normal and therefore, used for breeding programmes. During the last one and half years, blood samples were collected in the heparinized blood collecting tubes from 570 dairy bulls including 11 HF and 81 HF crossbred bulls for investigation of heterozygous/carrier animals for various genetic disorders, including Bovine Leukocyte Adhesion Deficiency Syndrome. The DNA extraction, PCR and RFLP were performed to find out carrier animals. PCR-RFLP techniques exhibited a polymorphism in CD18 gene in two Holstein crossbred bulls, which were confirmed by sequencing. The percentage of recessive allele in Holstein and Holstein crossbred samples was calculated to be 2.17. Rest all animals were found normal.

Development of a fast and economical genotyping protocol for bovine leukocyte adhesion deficiency (BLAD) in cattle.

Fast and economical means of assaying SNP’s are important in diagnostic assays, especially when a large number of animals have to be screened for a genetic disease. This study was aimed at the development of a fast and economical screening assay for bovine leukocyte adhesion deficiency (BLAD) which is an important genetic disease of cattle industry. Four primers were designed where the outer primers amplify a 354 bp amplicon of CD18 gene carrying the polymorphism responsible for BLAD. The specifically designed inner primers in conjunction with the modified reaction mixture and cyclic conditions ensured amplification of either of wild or mutated alleles. Together with outer primers, the inner primers generated typical banding pattern in agarose gel which discriminated the normal animal against the carrier. We successfully used this protocol in 200 bulls for genotyping the BLAD allele which confirmed by sequencing, showing a cent percentage concordance. With the developed assay the need for restriction digestion or use of costly equipment viz. real time PCR was eliminated. This genotyping assay ensured fast and economical genotyping and could be adopted in every laboratory with a minimum equipment requirement of thermocycler and gel documentation system.

Periodontal Involvement in Leukocyte Adhesion Deficiency: Review of the Literature and a Case Report

Objective: Leukocyte adhesion deficiency (LAD) is a rare, autosomal recessive inherited disorder; LAD-I which is the most common type, occurs due to mutations in the CD18 gene. This mutation down-regulates the expression of s2 integrin leukocyte cell surface molecules, which are necessary for the adhesion of leukocytes to endothelial cells, transendothelial migration, and chemotaxis. The major symptoms are recurrent severe bacterial infections without pus formation, recurrent or progressive necrotizing soft tissue infections, marked leukocytosis and severe progressive periodontitis accompanied by alveolar bone loss, periodontal pockets, and partial or total early loss of primary and permanent teeth. Cases: Herein, we report a case of moderate LAD-I in a nine year-old boy with severe alveolar bone loss and aggressive periodontitis. For several years, approximately every month, the patient was followed up to evaluate the status of primary and permanent dentition. During these visits, scaling and root planing were performed and severely mobile teeth were extracted. The patient is now under regular follow up. Conclusion: This case confirms the need for interactions between the medical groups to identify and manage medically compromised children with rare diseases. It is important to include LAD in the differential diagnoses of children presenting with periodontal disease. Early correct diagnosis of LAD has various benefits for patients.

Production and purification of high-titer foamy virus vector for the treatment of leukocyte adhesion deficiency

Compared to other integrating viral vectors, foamy virus (FV) vectors have distinct advantages as a gene transfer tool, including their nonpathogenicity, the ability to carry larger transgene cassettes, and increased stability of virus particles due to DNA genome formation within the virions. Proof of principle of its therapeutic utility was provided with the correction of canine leukocyte adhesion deficiency using autologous CD34+ cells transduced with FV vector carrying the canine CD18 gene, demonstrating its long-term safety and efficacy. However, infectious titers of FV-human(h)CD18 were low and not suitable for manufacturing of clinical-grade product. Herein, we developed a scalable production and purification process that resulted in 60-fold higher FV-hCD18 titers from ~1.7 × 104 to 1.0 × 106 infectious units (IU)/ml. Process development improvements included use of polyethylenimine-based transfection, use of a codon-optimized gag, heparin affinity chromatography, tangential flow filtration, and ultracentrifugation, which reproducibly resulted in 5,000-fold concentrated and purified virus, an overall yield of 19 ± 3%, and final titers of 1–2 × 109 IU/ml. Highly concentrated vector allowed reduction of final dimethyl sulfoxide (DMSO) concentration, thereby avoiding DMSO-induced toxicity to CD34+ cells while maintaining high transduction efficiencies. This process development results in clinically relevant, high titer FV which can be scaled up for clinical grade production.

461. Scale-Up and Manufacturing of High-Titer Foamy Virus Vector Containing Human CD18 for the Treatment of Leukocyte Adhesion Deficiency

Compared to other integrating viral vectors, foamy virus (FV) vectors have several distinct advantages as a gene transfer tool, including: the ability to carry larger expression cassettes, the increased stability due to a DNA genome formed in developing vector particles, and the random integration pattern without preference for promoter regions or active genes as is the case with gamma-retroviral and lentiviral vectors. In addition, the FV envelope has tropism for most cell types and is not associated with disease in humans. Combined, these properties make FV vectors the ideal candidate for gene therapy application. Proof of principle was provided by Bauer et al. (Nat. Med. 2008) showing cure of canine Leukocyte Adhesion Deficiency (LAD) using autologous CD34+ cells transduced with FV vector carrying the CD18 gene, without short or long-term adverse effects. Here we demonstrate the optimization of large scale manufacturing of FV vector carrying the human CD18 gene for the treatment of human LAD. As compared to the methods used in the canine LAD study, a 60-fold increase in FV-CD18 titer was observed using a combination of polyethylenimine (PEI) transfection and use of a codon-optimized gag, from approximately 1.70E+04 to 1.00E+06 Infectious Units/mL (IU/mL) on Raw 264.7 cells. An evaluation of purification strategies showed efficient vector purification and concentration of FV vector through a combination of heparin-affinity chromatography on the AKTA ready, a disposable fluid path chromatography unit, tangential flow filtration and ultracentrifugation, with an overall 5000-fold concentration and 20-30% recovery. This approach was compatible with large scale manufacturing in compliance with Good Manufacturing Practices (GMP). Although 5% DMSO is needed for optimal recovery of infectious FV vector from -80°C storage, highly concentrated vector can be diluted to reduce the DMSO concentration and toxicity to CD34+ cells, while maintaining effective transduction rates of up to 50%. This process is currently being finalized for clinical manufacturing in support of a Phase I trial for the treatment of LAD.Note: MN and DL equally contributed to this study.

Genetic risk of restenosis after percutaneous coronary interventions in the era of drug-eluting stents.

Our previous studies identified three single nucleotide polymorphisms (SNPs), rs419598 in the interleukin-1 receptor antagonist gene (IL1RN), rs235326 in the CD18 gene (ITGB2), and rs5918 in the platelet glycoprotein IIIa gene (ITGB3), as risk factors for restenosis after placement of bare metal stents in coronary arteries. The aim of the present study was to determine whether these SNPs remain risk factors after drug-eluting stent implantation. The study population included 2028 patients, who underwent drug-eluting stent implantation. The primary study endpoint was angiographic restenosis (diameter stenosis≥50% at the 6-month follow-up angiography) and clinical restenosis (target lesion revascularization at the 3-year follow-up). Angiographic restenosis was observed in 12.8% of the patients with genotype T/T, 14.6% with genotype T/C, and 13.0% with genotype C/C of IL1RN (P=0.60), in 13.1% of the patients with genotype C/C, 13.2% with genotype C/T, and 16.1% with genotype T/T of ITGB2 (P=0.53), and in 13.2% of the patients with genotype T/T, 14.8% with genotype T/C, and 9.6% with genotype C/C of ITGB3 (P=0.50). Clinical restenosis was present in 11.8% of the patients with genotype T/T, 12.5% with genotype T/C, and 9.9% with genotype C/C of IL1RN (P=0.63), in 13.3% of the patients with genotype C/C, 10.0% with genotype C/T, and 13.9% with genotype T/T of ITGB2 (P=0.07), and in 11.9% of the patients with genotype T/T, 12.1% with genotype T/C, and 10.2% with genotype C/C of ITGB3 (P=0.91). The SNPs rs419598 in IL1RN, rs235326 in ITGB2, and rs5918 in ITGB3, which have shown an association with restenosis after implantation of bare metal stents, were not related to restenosis after placement of drug-eluting stents.

CD11a, CD11c, and CD18 gene polymorphisms and susceptibility to Behçet's disease in Koreans.

Lesions of Behçet's disease (BD) show vascular infiltrates of immune cells expressing integrins. β2 integrins (CD11/CD18) play a major role in cell migration to the inflammatory lesion and also induce cytokine production. Thus, genetic polymorphisms of CD11/CD18 may be associated with the pathogenesis of BD. In this study, nine single nucleotide polymorphisms (SNPs) of the CD11a, CD11c, and CD18 were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and haplotype analysis in 305 BD patients and 266 healthy controls. The frequencies of genotype rs11574944 CC and haplotype rs11574944C-rs2230433G-rs8058823A in CD11a were significantly lower in BD patients. The frequencies of genotype rs2230429 CC, rs2929 GG, and haplotype rs2230429C-rs2929G in CD11c were higher in BD patients. The frequencies of genotype rs235326CC and haplotype rs2070946A-rs235326C-rs760456G-rs684G in CD18 were significantly higher in the BD patients than in the controls. Other SNPs in CD11a, CD11c, and CD18 gene were not significantly different. Therefore, the major genotype and haplotype of CD11a/CD18 may play a role in decreasing the susceptibility of BD, whereas the major genotype and haplotype of CD11c/CD18 may play a role in increasing the susceptibility of BD.

Genotyping of the Holstein-Friesian crossbred cattle for CD18 gene using PCR-RFLP

Genotyping of the Holstein-Friesian crossbred cattle for CD18 gene using PCR-RFLP