Background: Programmable nucleases are very promising tools of genome editing (GE), but they suffer from limitations including potential risk of genotoxicity which led to the exploration of safer ...approach of GE based on RNA-guided recombinase (RGR) platform. RNA-guided recombinase (RGR) platform operates on a typical recognition or target site comprised of the minimal pseudo-core recombinase site, a 5 to 6-base pair spacer flanking it and whole this central region is flanked by two guide RNA-specified DNA sequences or Cas9 binding sites followed by protospacer adjacent motifs (PAMs). Methods: The current study focuses on analysis of entire cattle genome to prepare a detailed map of target sites for RNA-guided hyperactivated recombinase Gin with spacer length six. For this, chromosome wise whole genomic sequence data was retrieved from Ensembl. After that search pattern for recombinase Gin with spacer length six was designed. By using this search pattern, RGR target sites were located by using dreg program of Emboss package. Result: Total number of RGR target sites identified in bovine genome for recombinase Gin was 677 with spacer length six. It was also investigated that whether these RGR target sites are present with in any gene or not and it was found that RGR target sites lies in both genic and intergenic region. Besides this, description of genes in context with these target sites was identified.
The present study was conducted to determine the effect of different cooling systems; Fan Fogger (FF) and Fan Pad (FP) on micro environment of poultry house, thermal comfort, welfare, egg production ...and egg quality parameters of laying hens. This experiment was conducted on 210, White Leghorn laying pullets (32 weeks old) during hot-dry summer months (May - July) under deep litter system of housing. The FP and FF cooling systems significantly dropped the mean shed temperature and increased the relative humidity. Thus, better THI resulted in increase in egg production by 4.66 % and 3.32 % under FP and FF systems over the control group. However, specific gravity, H.U, egg shell thickness, yolk index and yolk color were not significantly influenced by cooling treatments. Significantly lower levels of antioxidant enzymes viz. LPO, Catalase, G6PD, GPx and SOD was registered in cooling groups. Both the cooling devices contributed towards bird welfare by altering the behavioral expression from agonistic to non-agonistic activities.RNA-guided recombinases (RGR) are potentially valuable tools for basic research and genetic modifications. The platform has been demonstrated to do genome editing efficiently. The platform operates on a typical recognition site comprised of the degenerate recombinase site, a 5 to 6-base pair spacer flanking it and this whole central region is flanked by two guide RNA-specified DNA sequences or Cas9 binding sites which is followed by protospacer adjacent motifs. In present investigation, a detailed map of target sites for RNA-guided recombinase platforms based on hyperactivated recombinase Beta throughout the bovine genome was prepared. For this, Chromosome wise whole genomic sequence data was retrieved from Ensembl followed by designing search pattern for recombinase Beta with spacer length five. By using this search pattern, RGR target sites were located by using dreg program of Emboss package. In total,436 RGR target sites were identified in bovine genome for recombinase Beta with spacer length five. These RGR target site provide potential of being utilized for specific genomic integration, deletion or inversion.
In the present investigation, differentially expressed genes (DEGs) were studied using RNA sequencing (RNA-seq) technique in porcine peripheral blood mononuclear cells (PBMC) of weaned Ghurrah and ...crossbred piglets at 3-month age. Transcriptomic analysis was done using three different packages, namely, EBSeq, DESeq2, and edgeR, to identify the DEGs between Ghurrah and crossbred piglets. Total 7717 DEGs were commonly identified by all three packages, out of which 4151 genes found to be up-regulated, and 3566 genes were down-regulated. Functional annotation of these DEGs indicated metabolism as the most commonly enriched category followed by the immune response. Genes related to metabolism and growth were up-regulated in crossbred piglets as compared with Ghurrah piglets, whereas immunity-related genes were up-regulated in Ghurrah piglets elucidating the disease resistance nature of this indigenous breed over crossbred counterparts. Further, eight DEGs, namely,
LRP-1
,
ADCY4
,
ERRFI1
,
LDHD
,
ARG1
,
OASL
,
MGARP
, and
S100A8
, were validated by qRT-PCR in a separate set of biological samples and found to be in concordance with RNA-seq results. Finding in the present study provides insight into genes and their molecular mechanisms governing difference in growth performance between Ghurrah and crossbred pigs.
Classical swine fever is a highly contagious disease of pigs which courses from life-threatening to asymptomatic, depending on the virulence of the virus strain and the immune-competence of the host. ...The present study was undertaken to investigate the expression of immunologically important genes, viz. IFNα, IFNβ, SLA, SLA-2, SLA-DR, Ii, SLA-DM, CSK and JUN and to ascertain genetic group differences on the basis of humoral immune response. Blood samples were collected from 5 indigenous and 6 crossbred piglets at pre-vaccination and after 28th day of classical swine fever (CSF) vaccination. On 28th day, the competitive Enzyme Linked Immunosorbent Assay (cELISA) revealed poor humoral immune response (E2 antibodies) in indigenous piglets (84.80%) as compared to crossbred piglets (98.33%) in response to CSF vaccination. The expression level of genes was analyzed in three ways, viz. indigenous 28th day post-vaccination (28dpv) versus pre-vaccination, crossbred 28th day post-vaccination versus pre-vaccination and crossbred 28th day post-vaccination versus indigenous 28th day post-vaccination. The study showed that IFNα, IFNβ, SLA, SLA-2, Ii, SLA-DM, CSK and JUN were significantly upregulated in crossbred piglets than indigenous piglets at 28th day post-vaccination. But the SLA-DR was significantly downregulated in CSF vaccinated crossbred over indigenous piglets.
In present investigation, differential expression of transcriptome after classical swine fever (CSF) vaccination has been explored at the cellular level in crossbred and indigenous (desi) piglets. ...RNA Sequencing by Expectation-Maximization (RSEM) package was used to quantify gene expression from RNA Sequencing data, and differentially expressed genes (DEGs) were identified using EBSeq, DESeq2, and edgeR softwares. After analysis, 5222, 6037, and 6210 common DEGs were identified in indigenous post-vaccinated verses pre-vaccinated, crossbred post-vaccinated verses pre-vaccinated, and post-vaccinated crossbred verses indigenous pigs, respectively. Functional annotation of these DEGs showed enrichment of antigen processing-cross presentation, B cell receptor signaling, T cell receptor signaling, NF-κB signaling, and TNF signaling pathways. The interaction network among the immune genes included more number of genes with greater connectivity in vaccinated crossbred than the indigenous piglets. Higher expression of
IRF3
,
IL1β
,
TAP1
,
CSK
,
SLA2
,
SLADM
, and
NF-kB
in crossbred piglets in comparison to indigenous explains the better humoral response observed in crossbred piglets. Here, we predicted that the processed CSFV antigen through the T cell receptor signaling cascade triggers the B cell receptor-signaling pathway to finally activate MAPK kinase and NF-κB signaling pathways in B cell. This activation results in expression of genes/transcription factors that lead to B cell ontogeny, auto immunity and immune response through antibody production. Further, immunologically important genes were validated by qRT-PCR.
In the present study, the transcriptome profiling of peripheral blood mononuclear cells (PBMCs) of indigenous piglets against classical swine fever (CSF) vaccination was performed for elucidating the ...genetic basis of their differential humoral immunity. Piglets were vaccinated with lapinised strain of CSF virus (CSFV) followed by measurement of humoral immune response using c-ELISA at 28th day post vaccination (28dpv). The RNA sequencing data was analysed using established pipeline to determine set of differentially expressed genes (DEGs) in high responder as compared to low responder piglet. The differentially expressed important immune molecules were involved in regulating important pathways including antigen processing and presentation, T cell receptor signalling, B cell development, activation and signaling genes. The genes with differential expression also included TLR 3, 6, 7, 8, 9, and antiviral molecules such as MX, and ISG (Interferon stimulated genes) family members. The proteinprotein interaction of the immune genes was extracted for network representation. Most of the immune genes involved showed upregulation except the genes for antigen processing and presentation and T cell receptor signaling that were downregulated in the high responder. The immunologically important genes namely IFIT1, IFIT5, TAPBP, and TLR7 were validated using qRT-PCT and were observed to be in concordance with the RNA Seq results.
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