Transcript oct4 - SPUR
SPUR program 2007 Cloning of zebrafish genes involved in meiosis Carmen M. Rivera García Postlethwait Lab August 3rd , 2007 Different mechanisms of sex determination Genetic Sex chromosomes ? Environmental Temperature unknown XX XY humans turtles zebrafish Mechanism of sex determination unknown No sex chromosomes found Possible genetic sex determination influenced by environment ? Juvenile hermaphrodites with separate sexes in adults Zebrafish are juvenile hermaphrodites embryos juveniles adults Juvenile hermaphrodites females (Takahashi et al., 1977 Uchida et al., 2002) (ovaries) Gonad development ? males Oocytes in all juveniles Our GOAL: Understand the developmental processes that make the juvenile gonad to become ovary or testis (testes) Meiosis is an essential process during gonad development Our APPROACH: Study of meiosis in zebrafish gonads Meiosis in zebrafish female male 2n 2n (diploid) (diploid) ovaries testes Ovary section Testis section meiosis gonads egg meiosis sperm n n (haploid) (haploid) 2n (diploid) Experimental Strategies 1) Look for candidate genes: Search articles in Pubmed (NCBI) to find genes involved in meiosis in mouse 2) Selection of 4 genes to clone in zebrafish: oct4: octamer-4 sycp3: synaptonemal complex protein 3 atm: ataxia telangiectasia mutated dmc1: dosage suppressor of mck1 homolog Mouse genes involved in meiosis germ cell (gonads) oct4 oct4 is highly expressed in gonads. Is a transcription factor essential for the maintenance of totipotency in embryonic stem cells. (Bullejos and Koopman, 2004) Mouse genes involved in meiosis germ cell (gonads) oct4 enter meiosis oct4 When germ cells enter in meiosis oct4 expression is being extinguished (Bullejos and Koopman, 2004) Mouse genes involved in meiosis germ cell oct4 (gonads) enter meiosis oct4 formation of synaptonemal complex sycp3 recombination nodule Protein axes (lateral elements) sister chromatids sycp3 encodes a component of the synaptonemal complex, a meiosisspecific structure essential for synapsis of homologous chromosomes. (Dobson et al.,1994; Lammers et al., 1994) Mouse genes involved in meiosis germ cell oct4 (gonads) enter meiosis oct4 formation of synaptonemal complex sycp3 recombination nodule atm Protein axes (lateral elements) sister chromatids atm is associated with sites along the synaptonemal complex which are thought to be involved in meiotic recombination. (Plug AW et al., 1997) Mouse genes involved in meiosis germ cell oct4 (gonads) enter meiosis oct4 dmc1 is a meiosis-specific gene required to repair double-strand breaks and pairing between homologous chromosomes (Katrin et al,2004) formation of synaptonemal complex sycp3 recombination nodule dmc1 atm sister chromatids Protein axes (lateral elements) sister chromatids Experimental Strategies 3) Design of primers: mouse database blast zebrafish database (NCBI) (NCBI) Reciprocal blast should give us the original mouse gene Blast is a method to compare gene sequences Experimental Strategies 3) Design of primers: mouse database blast zebrafish database (NCBI) (NCBI) Reciprocal blast should give us the original mouse gene 4) Cloning of genes Gene nucleotide sequence from zebrafish Primer design for PCR PCR is a method to get multiple copies of our gene of interest Results Amplification of zebrafish meiotic genes by PCR oct4 100bp ladder O T sycp3 C- 100bp ladder O T atm C- 100bp ladder O T dmc1 100bp ladder C- O 600bp 600bp 600bp 700bp Fragment size: Fragment size: Fragment size: Fragment size: 778 bp 619 bp 601 bp 614 bp O = ovary T = testis C - = negative control Cutting out bands to clone genes C- Results Cloning of genes (I) Ligation of Bands and Transformation Plasmids E.coli cells (TOPO vector) ligation transformation PCR fragments (insert) Amp r Screening of colonies with insert Pick colonies Grow colonies in LB+Amp LB + Amp Cells grown in Amp plates ON 1h 37˚C PCR Results Cloning of genes (II) Selection of plasmids containing insert PCR using T7 and M13r primers 800bp 763 bp T7 M13r Insert 600bp Grow colonies that have plasmid+insert Plasmid with Insert LB + Amp Minipreps Sequencing of plasmids Shaker ON 37˚C Lysis of bacterial cells Recovering of plasmid oct4 Results Sequencing of plasmids using primers from vector to know insert orientation We have cloned oct4 gene from zebrafish blastn Clones sequenced using T7 and M13r primers 5’ 3’ Orientation of oct4 clones T7 oct4 T1 (#6.1) T7 oct4 T1 (#6.2) T7 oct4 O1(#7.1) T7 oct4 O1 (#7.2) atm Results Sequencing of plasmids using primers from vector to know insert orientation We have cloned atm gene from zebrafish blastn Clones sequenced using T7 primer 5’ 3’ Orientation of atm clones T7 T7 atm O1 (#5) atm O1 (#6) dmc1 Results Sequencing of plasmids using primers from vector to know insert orientation We have cloned dmc1 gene from zebrafish blastn Clones sequenced using T7 primer 5’ 3’ Orientation of dmc1 clones T7 T7 dmc1 T1 dmc1 T2 T7 dmc1 O1 T7 dmc1 T3 T7 T7 dmc1 O2 dmc1 O3 sycp3 Results Sequencing of plasmids using primers from vector to know insert orientation Have we cloned sycp3 gene from zebrafish ? blastn Clones sequenced using T7 primer Orientation of sycp3 clones T7 T7 scyp3 O1 (#10) scyp3 O1 (#9) T7 scyp3 T1 (#11) T7 scyp3 T1 (#12) Results Have we cloned sycp3 gene from zebrafish ? Putative zebrafish Sycp3 blast mouse database (NCBI) The gene that we have cloned is sycp3 Conclusions We have cloned 4 zebrafish genes with putative roles in meiosis Our results show that all cloned genes are expressed in adult ovary and testis in zebrafish Is the expression pattern novel in zebrafish? Is the expression pattern during meiosis novel in zebrafish? No No Yes atm No No Yes dmc1 No Yes Yes sycp3 Yes Yes Yes cloned zebrafish gene New zebrafish sequence ? oct4 Future work Analysis of expression of cloned genes by in situ hybridization dmc1 expression in adult zebrafish gonads Adult Ovary Adult testis 20X 20X Acknowledgments Postlethwait lab John Postlethwait Adriana Rodríguez-Marí Sara Wyckoff Ruth BreMiller and all people in the lab SPUR program Peter O’Day Beth Roy Summer Program for Undergraduate Research