Establishment And Identification Of SETD4 Gene Knockout Mice

In 2000,Stephen first discovered and reported the SET(suppressor of variegation,enhancer of zeste and trithora)domain family proteins.SET domain is named for its existence in the carboxyl terminal of three Drosophila genes:Suppressor of variegation(Su(var)3-9),Enhancer of zeste(E(z)),and Trithorax),SET domain structure is highly conserved in evolution,containing roughly 130 amino acid residues.We call such protein as SET domain-containing protein.Subsequent studies shown that SET domain-containing protein generally have histone and non-histone methyltransferase activity and play a role in substrate's methylation.Once histone methylated,the affinity of double-stranded DNA and histones will alter,which affects the loose or agglomerated state of chromatin,binding affinity of protein factors and chromatin,influences the capacity for transcription factors binding to DNA sequences.Different methylation sites reflect gene in transcriptional activation or inhibition.SET domain-containing protein involved in gene transcription regulation,heterochromatin formation,X chromosome inactivation,DNA damage repair and other major physiological functions via its methyltransferase activity.So once SET domain-containing proteins expression dysregulation,abnormal cell growth and ontogeny may happen,which leading to the occurrence and development of disease.More and more studies show that SET domain-containing proteins closely related to cancer and inflammation.SET domain family proteins such as Smyd2,can specifically facilitate H3K36 dimethylation at Tnf and Il6 promoters to suppress their transcription and inhibit NF-?B and ERK signaling,which suppresses the production of proinflammatory cytokines inducing by macrophage activation.Ezhl promotes TLR-triggered production of inflammatory cytokine including IL-6,TNF-? and IFN-? by suppressing the TLR negative regulator Tollip through its H3K27me3 methyltransferase activity.SETD3 takes part in the formation of B-cell lymphoma.Therefore,it's of great significance to study the function of SET domain family proteins for understanding the pathogenesis of inflammatory diseases and cancer.SETD4(SET domain-containing protein 4)is a new member of SET family,Searching the imformaton of setd4 gene on Ensemble Genomes database(http:www.ensemblgenomes.org/),it showed that murine setd4 gene located on mouse chromosome 16,93,583,457-93,604,063,total length of DNA is 20,606 bp.There are eight transcripts existing in setd4 gene,of which only two transcript Setd4-001(transcript ID:ENSMUST00000023669)and Setd4-201(transcript ID:ENSMUST00000113951)can be translated into SETD4 protein.Setd4-001 of full-length 1816 bp,Setd4-201 of full-length 1826 bp,both have 12 exons,of which the start sites and the length are the same except the exon 1 and exon 12.And the exon 2-11 are the encoding exon which play a role in encoding proteins.Therefore,Setd4-001 and Setd4-201 transcripts are encoded the same SETD4 protein of 439 amino acids,protein theoretical molecular size of 49 kDa.SETD4 is member of the SET family which also contains a highly conservative SET domain structure.It was presumed that SETD4 probably have histone and non-histone methyltransferase activity,participating in gene transcription regulation,heterochromatin formation,X chromosome inactivation,DNA damage repair and other major physiological functions like other SET family proteins.But there is less reports about SETD4,so we know little about its function.Only several papers suggests SETD4 may play a role in the development of breast cancer and HCC.The preliminary studies of our laboratory have found SETD4 had differential expression in liver cells of mice with endotoxin shock,which related to histone post-translational modifications.Further studies showed that SETD4 is distributed in the both of cytoplasm and nucleus of the RAW264.7 cell in the normal condition,but mainly in the cytoplasm.When following bacterial lipopolysaccharide(lipopolysaccharide,LPS)stimulation,SETD4 protein expression increased,as a result,the release of inflammatory cytokines enhanced.These suggest that SETD4 may involve in and play a positive role in the the regulation of LPS-mediated inflammatory cytokines.However,the actual situation in vivo will be more complex,whether the SETD4 protein still regulate inflammatory response in animals remains unclear.One of the most direct and effective measures is to knock out the target gene in mice in which circumstances we can detect the corresponding index of mice in disease models.For this purpose,we introduced the SETD4tm1a(KOMP)Wtsi heterozygous mice from the United States KOMP(Knockout Mouse Project)Repository.The construction strategy of this kind of SETD4 gene knockout mice was based on the Flp/FRT and Cre/LoxP system.Intron of setd4 gene was insertded with a gene disruption cassette which contains reporter gene,neomycin,Splice acceptor gene and was flanked with FRT sites.As to the target gene SETD4 exon 6,it was flanked with two LoxP sites.Through flexibly selecting Flp/FRT system or Cre/LoxP system can obtain either SETD4 conditional knockout mice or systemic knockout mice.Our study chose the EIIa-Cre mice which widely expressed the Cre recombinase,mating with SETD4tm1a(KOMP)Wtsi heterozygous mice.Cre recombinase can efficiently excised the target sequence between the two LoxP sites,so that we can obtain the SETD4 knockout mice which lays the foundation for our further study for the role of SETD4 in inflammation and its mechanism.According to these scientific problems,the experiment is divided into the following parts:The first part:Construction of SETD4 Knockout mice.SETD4tm1a(KOMP)Wtsi(SETD4flox/+)mice and EIIa-Cre mice were interbred,the offspring which is genotyping SETD4+/-.EIIa-Cre were crossed with C57BL/6 mice to obtain the mice with the SETD4+/-genotype,SETD4+/-heterozygous mice were inbred and then the SETD4-/-homozygous mice were gained.PCR was used to identify the genotype of the offspring.The seond part:Identification of SETD4 Knockout mice including phenotype identification and functional identification.The phenotype identification includes:DNA sequencing and BLAST aligment examed whether the exon 6 of SETD4-/-mice has been knock out.RT-PCR and qPCR detected the expression of SETD4 mRNA.of SETD4-/-mice.Western Blot detected the SETD4 protein expression of SETD4-/mice.The functional identification includes:isolated the peritoneal macrophages(pM(p)and bone marrow-derived macrophages(BMDMs)of SETD4-/-,SETD4+/+ mice,administering LPS stimulation at different time points.Quantitative PCR detected the changes of the mRNA expression.of TNF-? and IL-6 in BMDMs,then examed the release of cytokines in cell conditioned medium of pM? and BMDMs by liquidchip.In this way,we investigated whether SETD4 involved in the development of inflammation by regulating the release of cytokines.This thesis has made the experimental results as follows:First,PCR results showed genotypes of the offspring of SETD4 gene knockout mice in accordance with Setd4-/-.Second,we amplified the 699 bp PostCre positive band of SETD4-/-mice by PCR for DNA sequencing.The sequencing results blast with the sequence of SETD4 target allele in NCBI database,which showed that 1-326 bits of the tested sequences correspond to the 19873-20196 bits of SETD4 targeted allele(containing "LacZ","polyA" and "loxP" gene),292-668 bits of the tested sequences correspond to the 22787-23163 bits of SETD4 targeted allele(containing "loxP" gene and"3,arm"),and the target gene SETD4 exon 6 was not found.Third,we extracted the total RNA from Liver and Lung of SETD4-/-,SETD4+/+mice and detected the expression of SETD4 mRNA.In RT-PCR assay,SETD4+/+mice can amplify the 999 bp of SETD4 primer,but SETD4-/-mice can not.In quantitative PCR detection,compared with the wild type mice,expression of SETD4 mRNA in SETD4 gene knockout homozygous mice was significantly decreased.(n=3,P<0.001).Forth,we extracted the total protein from the BMDMs of SETD4-/-,SETD4+/+mice and detected its expression.The theoretical molecular weight of SETD4 was 49 kDa,but we found the samples of SETD4-/-,SETD4+/+ mice both can be detected bands at 44 kDa using SETD4(N-20)polyclonal antibody.Changing to SETD4(F-3)monoclonal antibody for examing,the bands at 44 kDa also both can be detected,however,we discovered a weak band at 49 kDa,which only can be examed in SETD4+/+sample.Fifth,we used the 293T cells which transfected with HA-SETD4 fusion protein to verify the specificity of SETD4 antibody.As the HA tag size is only 1 kDa,the positions of HA-SETD4 and SETD4 should be nearly at the same level.After successfully over-expressed in 293T cells,we found that the band of HA-SETD4 was dectected at about 49 kDa using HA antibody,while the bands of endogenous SETD4 examed by SETD4 antibody only at 44 kDa.The positions of these two bands are not roughly at the same level.In addition,endogenous SETD4 examed by SETD4 antibody also have a band at 49 kDa,but the band is relatively weak.Sixth,administerd the pMcp and BMDMs of SETD4-/-,SETD4+/+ mice with LPS stimulation and compared their release of cytokines.In SETD4 knock out mice,TNF-? mRNA level significantly decreased after LPS stimulation for 1 and the release of TNF-? also obviously reduced after LPS stimulation for 6 h and 12 h.And the IL-6 mRNA level significantly decreased after LPS stimulation for 3 h,the release of IL-6 obviously reduced after LPS stimulation for 6 h?12 h and 24 h,too.According to the experimental results,we can get the following preliminary conclusions:First:Based on the Cre/loxp system,SETD4flox/+ mice and EIIa-Cre mice were interbred,and then the offspring were crossed with C57BL/6 mice,can obtain the SETD4-/-homozygous mice.Second:In the DNA sequence of SETD4-/-mice,the target gene SETD4 exon 6 was not found.Compared with SETD4+/+ mice,the SETD4 mRNA level is significantly decreased.Western Blot result showed that there are existing differences between SETD4-/-mice and SETD4+/+ mice in 49 kDa which was the theoretical molecular weight of SETD4.All the evidences suggested that SETD4 gene knockout homozygous mice was successfully generated.Third:SETD4 protein has lower abundence in life,and the specificities of the existing SETD4 antibodies are poor.So,the target band is not easy to detect,and the non-specific bands was easily for us to make mistakes.Forth:SETD4 played a positive role in the early release of cytokine in the cell conditioned medium of pM? and BMDMs following LPS treatment,especially in the release of IL-6.In a word,we successfully generated SETD4 gene knockout homozygous mice which can be used for SETD4 function study.