Construction Of Targeting Vector For Mouse Naf1 Gene Knockout

Inflammatory response as a complicated process has an importantphysiological significance in vivo. Various inflammatory stimulators,including immune complex, virus, bacteria, tissue injury and so on, causethe process that leukocytes adhere to the vascular endothelial cells, crossvascular wall, generate and secrete various inflammatory media andchemokines, at the same time increase the quantity of partial blood streamand permeation of capillary vessel. A series of physiological andpathological processes trigger inflammatory response. The importantcharacters of inflammatory process are leukocyte adhesion,transendothelial migration of leukocytes and diapedesis. The essentialmolucular mechanism of this process is the interaction of leukocytes withvascular endothelial cells adhesion molecules. The adhesion of leukocytesand vascular endothelial cells in vivo is a process which includes theprimal adhesion of leukocytes rolling along the vascular wall, succedentlyfirm adhesion and transendothelial migration. Selectins on the surface ofvascular endothelial cells and selectin ligands on the surface of leukocytesmediate adhesion of leukocytes and vascular endothelial cells. The mostimportant one is P-selectin and its ligand PSGL-1.Naf1(Nef-associated factor 1) as the binding protein of Nef, whichwas cloned in 1999, is an important intracellular protein, expressed invarious human tissues and T-cell lines.The known functions of Naf1 arethat it interacted with HIV-1 matrix protein, then was packed into virusand influenced virus replication. The Naf1 gene generates two isoforms(Naf1α and Naf1β) containing four coiled-coil structures. The Naf1mRNA is ubiquitously expressed in human tissues and strongly expressedin peripheral blood lymphocytes and spleen. Naf1 overexpressionincreased cell surface CD4 expression, providing a novel mode of Nefaction in CD4 down-regulation. The known studies have shown Naf1 tobe a TNF-a inducible, disease-associated gene. A recent study has shownthat Naf1 can also bind the MAP kinase ERK2 and cause its retention inthe cytoplasm, which in turn leads to a reduction of ERK2 nuclearsignalling.We have constructed a vector as the bait for screening yeasttwo-hybrid libraries which was composed of 69 amino acids incytoplasmic domain of PSGL-1 and found a novel intracellular proteinNaf1. We hypothesize that PSGL-1 cross-linking by P-selectin mayrecruit p85 subunit by tyrosine phosphorylation Y552 in the YPPMsequence of Naf1, cause PI3K (phosphatidyl inositol-3 kinase) mediatingsignal transduction pathways and activate β2-integrins. It is reported thatNaf1 and Syk protein were concerned with the signal transductionpathways of NF-κB. Because of the important functions of Naf1 inmediating PSGL-1 signal transduction pathways, we successfullyconstructe mouse Naf1 targeting vector, designed based on the Cre-LoxPsystem and will further investigate the function of this gene by this geneby the gene knockout technology.First we searched the Naf1 genomic sequence in mouse genebank(NT₀96135.4), analyzed the exon and intron sequences and then foundthe beginning encoding sequences. The usual method was deleting partialor total targeting gene and relpacing the gene by positive-selectin cassettein order to have a clearly genetic background. Under usual condition thelength of deleted gene was 2～3kb and the fit length for vectorhomologous arms is 5～8kb in view of the influence of the length oftargeting vector homologous arms on targeting efficiency. At the sametime the two-side homologous arms of replacement targeting vector wereanisomerous, which were composed of shortarm and longarm. The lengthof shortarm was 0.5～2kb in order to be convenient for PCR screnningand the length of longarm was long enough in order to form chromosome-exchanging compound. Targeting vector need linearization beforetransfecting ES cell, so there were no less than one single restrictive siteon the outside of homologous arms to linearize the targeting vector.We confirm that the deletion domain is 1.4kb length including exon3and exon4. The upstream and downstream sequences of this domain weresearched and analyzed the restrictive sites. The construction strategy oftargeting vector is using the 1.3kb upstream exon3 as shortarm and 5.4kbdownstream exon4 as longarm. Longarm was separated into two partsbased on the gene sequences and restrictive sites. Three pairs of specificmatching primers were designed according to Naf1 genomic sequences.The target gene was amplified by polymerase chain reaction (PCR)technique. The reaction system is: template genomic DNA 1.5ul, 10×LAPCR buffer 5ul,dNTP Mixture 10mol/L 2ul, primers 20mol/L 1.5ul each,LA Taq (5U/ul) 0.5ul and ddH2O to 37.5ul. The condition of PCR reactionconsisted of denaturing for 6min at 94℃, pause at 5.5min and adding toLA Taq (5U/ul) 0.5ul, 35 cycles of denaturing for 1min at 94℃, annealingfor 1min at 65℃ and extending for 3min(according to gene length1kb/min) at 72℃, and extending for 5min at 72℃. Finally, the PCRproduct was identified by 1% agarose gel electrphoresis. If the result wasnot very clear, we could regulate annealing temperature until getting theclearer one.The PCR product was recovered and inserted into pMD18-T vector(TaKaRa). The mole proportion of vector and insert DNA was 1:2～10.The ligation reaction system was pMD18-T vector(50ng/ul)1ul, InsertDNA 4.5ul and Ligation Solution I 5ul. The mixed system was incubatedin water bath at 16℃ over night in order to form recombinant. Therecombinant was transformed into the bacterial strain E.Coli DH5αaccording to routine protocol that 10ul recombinant and 100ul competentDH5α as host cells were mixed. 100ul transformed cells was spreaded onan LB plate containing ampicillin (100ug/ml), X-gal (50mg/ml) and IPTG(20mg/ml) and then incubated at 37 ℃ over night. The positiverecombinant clone was screened by blue-white test. The correctrecombinant plasmids were confirmed by PCR reaction and sequencedusing RV-M/M13 as primer. The sequencing result of TA-clone wascompared with genebank sequence in order that the mutant ratio was lessthan 0.5%.We used pBluescriptII KS+ as targeting vector backbone. Accordingto the restrictive sites of Naf1 and vector, the project was designed thatshortarm was inserted by XbaI and SmaI, neo cassette by SmaI and ClaI,longarmA by ClaI and EcoRI and longarmB by EcoRI and XhoI. AnEcoRI was mutant in front of the SalI in pBluescriptII KS+. Then neocassette, shortarm, longarmA and longarmB were inserted into vectorrespectively. Finally, the correct recombinant plasmids were confirmed bydouble restrictive enzyme cleavage as well as sequencing. Wesuccessfully finished the construction of Naf1 gene targeting vector.