Molecular and genetic aspects of technology of generation transgene mice with the integrated human of Nacetyltransferase genes (NAT1 and NAT2)

The DNA constructions with nucleotide sequences of human genes of NAT1 and NAT2 and promoter-enhancedregion of a mice albumin gene were created. The coding parts of NAT1 and NAT2 genes were amplified from a matrix of genomic human DNA with use of NAT1-Not and NAT1-Xho oligonucleotides in case of NAT1 and NAT2-Not and NAT2-Xho in case of NAT2 . Plasmids of pSI-NAT1 and pSI-NAT2 split by BamHI and EcoRV endonuclease (the site of a restriction of EcoRV was entered in enAlb-F oligonucleotide). Synthesis of the oligonucleotide primers and detection of human genes expression and the created transgene animals with the use of specific oligonucleotide primers was carried out in parallel. At the following stages allocation of DNA/RNA from blood plasma test, amplification of genomic DNA, identification of PCR products was carried out by method of a horizontal electrophoresis. Existence in gel of DNA strip corresponding size testified to existence in a sample of a required gene. Transgene mice (F0) with the designs integrated into their genome including nucleotide sequences of genes of human NAT1 and NAT2 under the albumin gene mice promoter were generated. Total effectiveness of a transgenesis for NAT1 gene is 1,1% and for NAT2 - 0,6%. The analysis of integration of transgenes in various descendants (F1) organs and tissues of primary transgene mice (F0) showed that human genes NAT1 and NAT2 was found in the cells of all three germinal leaves.

DNA constructions NAT1 and NAT2 genes, promoter-enhanced region of gene, plasmids, high-specific primers, detection of a genes expression, amplification, transgenesis

1. Езерский В.А., Тевкин С.И., Трубицина Т.П., Колоскова Е.М., Шишиморова М.С., Безбородова О.А., Якубовская Р.И., Рябых В.П. Интеграция и тканеспецифическая экспрессия гена лактоферрина человека в молочной железе трансгенных кроликов // Проблемы биологии продуктивных животных. 2013. № 4. С. 33-52.

2. Каркищенко Н.Н., Рябых В.П., Каркищенко В.Н., Колоскова Е.М. Создание гуманизированных мышей для фармакотоксилогических исследований (успехи, неудачи и перспективы) // Биомедицина. 2014. № 3. С. 4-22.

3. Каркищенко Н.Н., Петрова Н.В., Слободенюк В.В. Высокоспецифичные видовые праймеры к генам NAT1 и NAT2 для сравнительных исследований у человека и лабораторных животных // Биомедицина. 2014. № 2. С. 4- 17.

4. Blin N., Stafford D.W. A general method for isolation of high molecular weight DNA from eukaryotes // Nucleic acids res. 1976. No. 3(9). Pp. 2303-8.

5. Boukouvala S., Fakis G. Arylamine N-acetyltransferases: what we learn from genes and genomes // Drug metabolism reviews. 2005. No. 37. Pp. 511-564.

6. Butcher N.J., Arulpragasam A., Goh H.L., Davey T., Minchin R.F. Human arylamine N-acetyltransferase-1 interacts with EHZF, a multi-functional transcription co-factor // In: Third International Workshop on Arylamine N-acetyltransferases. -Vancouver, Canada. 2004.

7. Cao W., Chau B., Hunter R., Strnatka D., McQueen C.A., Erickson R.P. Only low levels of exogenous N-acetyltransferase can be achieved in transgenic mice // Pharmacogenetics J. 2005. No. 5. Pp. 255-261.

8. Cornish V.A., Pinter K., Boukouvala S., Johnson N., Labrousse C., Payton M., Priddle H., Smith A.J., Sim E. Generation and analysis of mice with a targeted disruption of the arylamine N-acetyltransferase type 2 gene // Pharmacogenomics J. 2003. No. 3. Pp. 169-177.

9. Coughtrie M.W.H., Sharp S., Maxwell K., Innes N.P. Biology and function of the reversible sulfation pathway catalysed by human sulfotransferases and sulfatases // Chem.-biol. interact. 2003. No. 109. Pp. 3-27.

10. Erickson R., Morgan C., McQueen C.A. 2nd International NAT Workshop. - Eynsham, Oxford. 2000. Abstract. No. 1.

11. Green M.D., Tephly T.R. Glucuronidation of amine substrates by purified and expressed UDP-glucuronosyltransferase proteins // Drug metab. dispos. 1998. No. 26. Pp. 860-867.

12. Karolyi J., Erickson R.P., Liu S. Genetics of susceptibility to 6-aminonicotinamide-induced cleft palate in the mouse: studies in congenic and recombinant inbred strains // Teratology. 1988. No. 37. Pp. 283-287.

13. Karolyi J., Erickson R.P., Liu S., Killewald L. Major effects on teratogen-induced facial clefting in mice determined by a single genetic region // Genetics. 1990. No. 126. Pp. 201-205.

14. Leff M.A., Epstein P.N., Doll M.A., Fretland A.J., Devanaboyina U.S., Rustan T.D., Hein D.W. Prostate-specific human N-acetyltransferase 2 (NAT2) expression in the mouse // J. Pharmacol. exp. ther. 1999. No. 290(1). Pp. 182-187.

15. Nebert D.W. Polymorphisms in drug metabolising enzymes: what is their clinical relevance and why do they exist? // Am. J. hum. genet. 1997. No. 60. Pp. 265-271.

16. Nelson D.R., Koymans L., Kamataki T., Stegeman J.J., Feyereisen R., Waxman D.J., Waterman M.R., Gotoh O., Coon M.J., Estabrook R.W., Gunsalus I.C., Nebert D.W. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature // Pharmacogenetics. 1996. No. 6. Pp. 1-42.

17. Sim E., Pinter K., Mushtaq A., Upton A., Sandy J., Bhakta S., Noble M. Arylamine N-acetyltransferases: a pharmacogenomic approach to drug metabolism and endogenous function // Biochem. soc. trans. 2003. No. 31. Pp. 615-619.

18. Sugamori K.S., Wong S., Brenneman D., Lu X., Grant D.M. NAT3 knockout or over expression in mice has no influence on N-acetylation activity // Drug metab. rev. 2004. No. 36. 164 p.

19. Sugamori K.S., Wong S., Gaedigk A., Yu V., Abramovici H., Rozmahel R., Grant D.M. Generation and functional characterization of arylamine N-acetyltransferase Nat1/Nat2 double-knockout mice // Mol. pharmacol. 2003. No. 64. Pp. 170-179.

20. Wong L.-L. Cytochrome P450 monooxygenases // Curr. opin. chem. biol. 1998. No. 2. Pp. 263-268.