Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
 2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A high-copy-number plasmid, pLink, was constructed to allow the direct selection in Escherichia coli of a neo fusion gene capable of conferring Geneticin (G418) resistance on mouse L cells. pLink was derived from pdMmtneo by insertion of a KpnI linker within the 5'-coding region of the neo gene. This created a minus-one frameshift mutation resulting in a translational termination within the N-terminal region of the protein. The Neo activity was restored by insertion into the modified neo gene of a piece of coding sequence derived from human HPRT cDNA. The resulting plasmid, pAH, was microinjected into mouse A9 cells and shown to confer resistance to G418.
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PMID:Construction of a neo fusion gene for expression in both prokaryotic and eukaryotic cells. 218 89

Unique, small sequences (sequence tag sites) have been identified at the 3' ends of most human genes that serve as landmarks in genome mapping. We investigated whether a single copy gene could be isolated directly from total human DNA by transformation-associated recombination (TAR) cloning in yeast using a short, 3' unique target. A TAR cloning vector was constructed that, when linearized, contained a small amount (381 bp) of 3' hypoxanthine phosphoribosyltransferase (HPRT) sequence at one end and an 189-bp Alu repeat at the other end. Transformation with this vector along with human DNA led to selective isolations of the entire HPRT gene as yeast artificial chromosomes (YACs) that extended from the 3' end sequence to various Alu positions as much as 600 kb upstream. These YACs were retrofitted with a NeoR and a bacterial artificial chromosome (BAC) sequence to transfer the YACs to bacteria and subsequently the BACs to mouse cells by using a Neo selection. Most of the HPRT isolates were functional, demonstrating that TAR cloning retains the functional integrity of the isolated material. Thus, this modified version of TAR cloning, which we refer to as radial TAR cloning, can be used to isolate large segments of the human genome accurately and directly with only a small amount of sequence information.
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PMID:Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3' end target sequence. 953 61

Using the HPRT genomic DNA fragment and synthesized oligonucliotides, pSP-HPRT-F-Neo-F3 was designed and constructed as a replacement gene targeting vector by usual molecular cloning techniques. Structure of pSP-HPRT-F-Neo-F3 was identified by restrictive digestion analysis and partly sequencing. Then linearized pSP-HPRT-F-Neo-F3 DNA was electroporated into ES cells, and transfected cells were screened by being cultured in medium containing 200 micrograms/mL G418 and 2 micrograms/mL 6-GT. Twenty-four double drug resistant clones were picked up and analyzed, among them, two clones were proved to have taken place the required recombination by PCR and southern blotting analysis.
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PMID:[Construction of a Flp "exchange cassette" contained vector and gene targeting in mouse ES cell]. 1179 23

Exchange plasmid pF-HPRT-F3 and Flp expression plasmid pCMV-Flp were constructed and then introduced using electroporation system into F18 ES cell line, which have an exchange cassette F-Neo-F3 at HPRT locus. After HAT selection, HAT resistant clones were obtained. Then G418 sensitivity test and Southern blotting were carried out to screen RMCE recombinants. The results indicated that RMCE had taken place in three of 12 HAT resistant clones. The frequency is 25%. The result demonstrates that it is realizable to introduce transgene to HPRT locus by using Flp recombinase mediated cassette exchange reaction.
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PMID:[The study of a HPRT locus-specific transgenic method based on FLP recombinase mediated cassette exchange]. 1254 6

The 3.0 kb 5' arm (long arm, LA) of rat HPRT gene knock-out vector was cut by Sal I from rat HPRT gene genomic bacterial artificial chromosome (BAC) , and the 1.7 kb 3' arm (short arm, SA) was proliferated by PCR. Neo', 5' arm, 3' arm were sequentially cloned into pBS vector's relative restriction enzyme sites. For acquirement of tk gene, the 5' arm -Neo' -3' arm fragment inserted into pKO vector to construct pKO-HPRT. The pKO-HPRT was linearized by Not I, extracted by ethidium bromide, butanol and phenol/chloroform, and dialyzed by 0.025 microlmol/L Millipore. At the same time, rat neural stem cells cultured from E14.5-16.5 rat fetal brain. Passage 2 rFNSCs was tranfected by linearized pKO-HPRT with Fugene-6t transfection reagents. After 80 microg/ml G418 and 0.2 micromol/L ganciclovir selection, the survived cells was cultured in suspension to form neural spheres. The spheres can be picked up under the microscopy, and proliferated in 96- ,48- and 24-well plates sequentially. When the cell number reached 4 x 10(3)/well, half cells was lysed by lysis buffer to extract DNA, the other half was kept on growing to freeze and extract RNA. The knock-out cell colonies first detected by PCR, then confirmed by Southern blot and RT-PCR. All the results show that we have knocked out HPRT gene in three rat fetal neural stem cell colonies from 32 colonies.
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PMID:[HPRT gene knock-out from rat fetal neural stem cells]. 1546 19

Prader-Willi syndrome (PWS) is a neurogenetic disorder caused by loss of paternally expressed genes on chromosome 15q11-q13. The PWS-critical region (PWScr) contains an array of non-protein coding IPW-A exons hosting intronic SNORD116 snoRNA genes. Deletion of PWScr is associated with PWS in humans and growth retardation in mice exhibiting ~15% postnatal lethality in C57BL/6 background. Here we analysed a knock-in mouse containing a 5'HPRT-LoxP-Neo(R) cassette (5'LoxP) inserted upstream of the PWScr. When the insertion was inherited maternally in a paternal PWScr-deletion mouse model (PWScr(p-/m5'LoxP)), we observed compensation of growth retardation and postnatal lethality. Genomic methylation pattern and expression of protein-coding genes remained unaltered at the PWS-locus of PWScr(p-/m5'LoxP) mice. Interestingly, ubiquitous Snord116 and IPW-A exon transcription from the originally silent maternal chromosome was detected. In situ hybridization indicated that PWScr(p-/m5'LoxP) mice expressed Snord116 in brain areas similar to wild type animals. Our results suggest that the lack of PWScr RNA expression in certain brain areas could be a primary cause of the growth retardation phenotype in mice. We propose that activation of disease-associated genes on imprinted regions could lead to general therapeutic strategies in associated diseases.
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PMID:Maternal transcription of non-protein coding RNAs from the PWS-critical region rescues growth retardation in mice. 2684 93