To establish stable cell lines that produce recombinant multisubunit proteins, it is usually necessary to cotransfect cells with several independent gene constructs. Here, we show that a stepwise fusion of individually transfected cells, results in a fused cell-line that secretes a complete multisubunit protein. Functional expression of recombinant multisubunit proteins may require a deﬁned expression ratio between each protein subunit. The cell-fusion technology described allows a predeﬁned expression level of each subunit.
Loss of tumor suppressors contributes to numerous cancer types. Many, but
not all, proteins encoded by tumor suppressor genes have antiproliferative activity
and halt cell-cycle progression. In this chapter, we present three methods
that have been utilized to monitor the antimitogenic action exerted by tumor
suppressors. Tumor suppressor function can be demonstrated by colony formation
assays and acquisition of the flat-cell phenotype.
Two H7721 human hepatocarcinoma cell lines showing moderate and high
expression of a1,3-fucosyltransferase (FucT)-VII cDNA were established
and designated FucTVII-M and FucTVII-H, respectively. In a1,3-FucT-VII-transfected cells, expression of insulin receptor (InR)a- and bsubunits
and epidermal growth factor receptor (EGFR) on the cell surface and in
cells, as well as the sialyl Lewis X (SLe
, the product of a1,3-FucT-VII)
content of the EGFR were unchanged.
The red cell intercellular adhesion molecule-4 (ICAM-4)
binds to different members of the integrin receptor families.
Tobetterdefine the ICAM-4 integrinreceptor specificity, cell
transfectants individually expressing various integrins were
used to demonstrate thataLb2,aMb2,andaIIbb3(activated)
bind specifically and dose dependently to the recombinant
ICAM-4-Fcprotein.We also showthat cell surface ICAM-4
interacts with the cell surface aVb3
Asheat shockproteins (Hsps) are involved inprotecting cells
and also in the pathophysiology of diseases such as inflam-mation, cancer and neurodegenerative disorders, modula-tors ofHsp expression inmammalian cells would seemto be
useful for the treatment of various diseases. In this study, we
isolated mammalian cell lines for screening of Hsp modu-lators; mouse C3H10T1/2 cells stably transfected with a
plasmid containing the mouse Hsp105 or human Hsp70B
promoter upstream of a luciferase or b-galactosidase
reporter gene, respectively....
There is extensive evidence that FcR c-chain couples to the collagen receptor glycoprotein VI (GPVI) and becomes phosphorylated on tyrosines upon receptor cross-linking. However, it is not established whether this receptor complex is suﬃcient to initiate the signalling cascade. We transfected GPVI and the FcR c-chain into the human erythroleukaemia cell line K562, which lacks detectable expression of GPVI and the FcR c-chain. The results show that GPVI is unable to signal when expressed alone, despite its surface expression, upon stimulation with the snake C-type lectin, convulxin....
Madin–Darby canine kidney (MDCK) cells, which do not normally express
the proteoglycan (PG) serglycin, were stably transfected with cDNA for
human serglycin fused to a polyhistidine tag (His-tag). Clones with differ-ent levels of serglycin mRNA expression were generated. One clone with
lower and one with higher serglycin mRNA expression were selected for
Expression of DNase II in macrophages is potentially cru-cially important in the removal of unwantedDNA.We have
previously shown that DNase II expression is up-regulated
at the transcriptional level during the phorbol 12-myristate-13-acetate (PMA)-induced differentiation of HL-60 and
THP-1 cells. In this study, we investigated thecis-regulatory
elements and transcription factors involved in this process in
A Drosophila melanogaster cDNA clone (GH01916)
encoding a putative 723-residue long (82 kDa) protein
(CG 7415) and displaying 50% identity with mammalian
cytosolic dipeptidyl aminopeptidase (DPP) III was func-tionally expressed in Schneider S2
cells. Immunocytochemi-cal studies using anti-(rat liver DPP III) Ig indicated the
expression of this putative DPP III at the outer cell mem-brane and into the cytosol of transfected cells.
Peptides which should be generated from the neuropeptide
FF (NPFF) precursor were identified in a neuronal (human
neuroblastoma SH-SY5Y) cell line and in COS-7 cells after
transient transfection of the human proNPFFAcDNA and
were comparedwith those detected in themouse spinal cord.
After reverse-phase high performance liquid chromatogra-phy of soluble material, NPFF-related peptides were im-munodetected with antisera raised against NPFF and
identified by using on-line capillary liquid chromatography/
nanospray ion trap tandem mass spectrometry. ...
Cyclin-dependent kinase-5 (Cdk5) is a serine/threonine
kinase activated by its neuron-specific activator, p35, or its
truncated form, p25. It has been proposed that the deregu-lation of Cdk5 activity by association with p25 in human
brain tissue disrupts the neuronal cytoskeleton and may be
involved in neurodegenerative diseases such as Alzheimer’s
Research on ion channels has exploded in the last few decades and it is now
clear that ion channels play essential roles in cell biology and physiology, with
their dysfunction being the root cause of many human diseases. Understanding
human biology in the post-genome sequencing era requires that the function of
the protein products encoded by these recently sequenced genes be quantified.
The third section of this book is ‘Adeno-associated-virus Vector’. Chapter nine by Sun
et al. introduces the adeno-associated virus (AAV) mediated β-thalassemia gene
therapy. Human hematopoietic stem cells (HSCs) were obtained from β-thalassemia
patients, transfected with the recombinant AAV containing β-globin gene. The
transfected cells were then transplanted into Nude/SCID mice, and the long term
expression of β-globin in vivo was examined. In the tenth chapter, Korecka et al.
compare AAV serotypes for gene delivery to dopaminergic neurons in the substantia
The CD53 antigen is a member of the tetraspanin membrane protein family that is expressed in the lymphoid-myeloid lineage. We have studied the implication of CD53 antigen in signal transduction by determining the eﬀect of its ligation on the c-Jun N-terminal kinase (JNK) in diﬀerent cell types. Ligation of the rat or human CD53 antigen induces a threeto fourfold transient activation of JNK activity that peaks at 3–5 min. The eﬀect was detected by assaying the endogenous or exogenous (transfected) JNK activity....
Gene transfer into many cell types has been successfully used to develop alternative and adjunct approaches to conventional medical treatment. However, effective transfection of postmitotic neurons remains a challenge. The aim of this study was to develop a method for gene transfer into rat primary dorsal root ganglion neurons using sonoporation. Methods: Dissociated cells from adult rat dorsal root ganglion (DRG) cells were sonicated for 1-8 s at 2.5-10 W to determine the optimal ultrasound duration and power for gene transfection and cell survival.
Tuyển tập báo cáo các nghiên cứu khoa học quốc tế ngành hóa học dành cho các bạn yêu hóa học tham khảo đề tài: Comparing the immunosuppressive potency of naïve marrow stromal cells and Notch-transfected marrow stromal cells
RNA-mediated gene silencing (RNA interference) is a powerful way to
knock down gene expression and has revolutionized the fields of cellular
and molecular biology. Indeed, the transfection of cultured cells with small
interfering RNAs (siRNAs) is currently considered to be the best and easi-est approach to loss-of-function experiments.