Enrichment of nucleofected primary human CD4+ T cells: A novel and efficient method for studying gene function and role in human primary T helper cell differentiation

Tahvanainen, Johanna; Pykäläinen, Maritta; Kallonen, Teemu; Lähteenmäki, Hanna; Rasool, Omid; Lahesmaa, Riitta

doi:10.1016/j.jim.2005.11.024

Cited by 30 publications

(29 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The Gateway Vector Conversion System (Invitrogen) was used to modify pIRES2-H-2K k vector (26) Nucleofection, dead cell removal, and enrichment of transfected cells…”

Section: Western Blottingmentioning

confidence: 99%

Activating Transcription Factor 3 Is a Positive Regulator of Human IFNG Gene Expression

Filén

Ylikoski

Tripathi

et al. 2010

The Journal of Immunology

Self Cite

View full text Add to dashboard Cite

IL-12 and IL-18 are essential for Th1 differentiation, whereas the role of IFN-α in Th1 development is less understood. In this microarray-based study, we searched for genes that are regulated by IFN-α, IL-12, or the combination of IL-12 plus IL-18 during the early differentiation of human umbilical cord blood CD4+ Th cells. Twenty-six genes were similarly regulated in response to treatment with IL-12, IFN-α, or the combination of IL-12 plus IL-18. These genes could therefore play a role in Th1 lineage decision. Transcription factor activating transcription factor (ATF) 3 was upregulated by these cytokines and selected for further study. Ectopic expression of ATF3 in CD4+ T cells enhanced the production of IFN-γ, the hallmark cytokine of Th1 cells, whereas small interfering RNA knockdown of ATF3 reduced IFN-γ production. Furthermore, ATF3 formed an endogenous complex with JUN in CD4+ T cells induced to Th1. Chromatin immunoprecipitation and luciferase reporter assays showed that both ATF3 and JUN are recruited to and transactivate the IFNG promoter during early Th1 differentiation. Collectively, these data indicate that ATF3 promotes human Th1 differentiation.

show abstract

“…The Gateway Vector Conversion System (Invitrogen) was used to modify pIRES2-H-2K k vector (26) Nucleofection, dead cell removal, and enrichment of transfected cells…”

Section: Western Blottingmentioning

confidence: 99%

Activating Transcription Factor 3 Is a Positive Regulator of Human IFNG Gene Expression

Filén

Ylikoski

Tripathi

et al. 2010

The Journal of Immunology

Self Cite

View full text Add to dashboard Cite

show abstract

“…The NDFIP2 shRNA plasmid construct, targeting (5Ј-GAGGAAGAGTGT CCACCAAGA-3Ј), was generated by cloning the NDFIP2 shRNA oligonucleotide into the BglII and XhoI sites of the previously modified pSuper-H2K-pIRES2 plasmid, which contains a truncated H2K cell surface selection marker (18). Similarly, a pSuper-H2K-pIRES2-scramble1-shRNA, (5Ј-AATT CTCCGAACGTGTCACGT-3Ј), was designed.…”

Section: Short-hairpin Rna (Shrna) Mediated Gene Knockdown During Thementioning

confidence: 99%

“…In addition, two synthetic small interfering RNA oligos (one of which targeting the same sequence as the NDFIP2 shRNA shown above and the other targeting the 5Ј-CUGGAUAU UUCAAUGGACAUU-3Ј NDFIP2 sequence; Sigma-Aldrich) were used to knock down NDFIP2. For the STAT6 knockdown studies, the previously prepared pSuper-H2K-STAT6-shRNA plasmid, targeting (5Ј-GAATCAGTCA ACGTGTTGTCAG-3Ј), and the pSuper-H2K-scramble2-shRNA (5Ј-GCGC GCTTTGTAGGATTCG-3Ј) were used (18). Furthermore, another pSuper-H2K-STAT6-shRNA plasmid, targeting 5Ј-CAGTTCCGCCACTTGCCAAT-3Ј, was used in one replicate culture.…”

Section: Short-hairpin Rna (Shrna) Mediated Gene Knockdown During Thementioning

confidence: 99%

“…Furthermore, another pSuper-H2K-STAT6-shRNA plasmid, targeting 5Ј-CAGTTCCGCCACTTGCCAAT-3Ј, was used in one replicate culture. Cell transfections, dead cell removal, enrichments (Ͼ98%), and differentiations were performed as recently described (18). The cells were activated with anti-CD3 plus anti-CD28 and were induced to polarize to Th1 and Th2 direction as described above.…”

Section: Short-hairpin Rna (Shrna) Mediated Gene Knockdown During Thementioning

confidence: 99%

See 1 more Smart Citation

Genome-Wide Identification of Novel Genes Involved in Early Th1 and Th2 Cell Differentiation

Lund

Löytömäki

Naumanen

et al. 2007

The Journal of Immunology

Self Cite

View full text Add to dashboard Cite

Th cell subtypes, Th1 and Th2, are involved in the pathogenesis or progression of many immune-mediated diseases, such as type 1 diabetes and asthma, respectively. Defining the molecular networks and factors that direct Th1 and Th2 cell differentiation will help to understand the pathogenic mechanisms causing these diseases. Some of the key factors regulating this differentiation have been identified, however, they alone do not explain the process in detail. To identify novel factors directing the early differentiation, we have studied the transcriptomes of human Th1 and Th2 cells after 2, 6, and 48 h of polarization at the genome scale. Based on our current and previous studies, 288 genes or expressed sequence tags, representing ∼1–1.5% of the human genome, are regulated in the process during the first 2 days. These transcriptional profiles revealed genes coding for components of certain pathways, such as RAS oncogene family and G protein-coupled receptor signaling, to be differentially regulated during the early Th1 and Th2 cell differentiation. Importantly, numerous novel genes with unknown functions were identified. By using short-hairpin RNA knockdown, we show that a subset of these genes is regulated by IL-4 through STAT6 signaling. Furthermore, we demonstrate that one of the IL-4 regulated genes, NDFIP2, promotes IFN-γ production by the polarized human Th1 lymphocytes. Among the novel genes identified, there may be many factors that play a crucial role in the regulation of the differentiation process together with the previously known factors and are potential targets for developing therapeutics to modulate Th1 and Th2 responses.

show abstract

“…These nonviral methods of gene transfer into primary human T cells (Tahvanainen et al, 2006;Magg et al, 2009) and HSCs (Sumiyoshi et al, 2009) are currently being developed and tested in a growing number of in vitro studies using gene markers. Some of the methods have shown good transfection efficiency and expression levels.…”

Section: Nonviral Methods Of Gene Transfermentioning

confidence: 99%

Contributions of Gene Marking to Cell and Gene Therapies

Barese

Dunbar²

2011

Human Gene Therapy

View full text Add to dashboard Cite

show abstract

Enrichment of nucleofected primary human CD4+ T cells: A novel and efficient method for studying gene function and role in human primary T helper cell differentiation

Cited by 30 publications

References 27 publications

Activating Transcription Factor 3 Is a Positive Regulator of Human IFNG Gene Expression

Activating Transcription Factor 3 Is a Positive Regulator of Human IFNG Gene Expression

Genome-Wide Identification of Novel Genes Involved in Early Th1 and Th2 Cell Differentiation

Contributions of Gene Marking to Cell and Gene Therapies

Contact Info

Product

Resources

About