Editing Xenopus (section)

=== Investigation of human disease genes ===

All modes of ''Xenopus'' research (embryos, cell-free extracts, and oocytes) are commonly used in direct studies of human disease genes and to study the basic science underlying initiation and progression of cancer.<ref>{{cite journal | vauthors = Hardwick LJ, Philpott A | title = An oncologist׳s friend: How Xenopus contributes to cancer research | journal = Developmental Biology | volume = 408 | issue = 2 | pages = 180–187 | date = December 2015 | pmid = 25704511 | pmc = 4684227 | doi = 10.1016/j.ydbio.2015.02.003 | series = Modeling Human Development and Disease in Xenopus }}</ref> ''Xenopus'' embryos for ''in vivo'' studies of human disease gene function:   ''Xenopus'' embryos are large and easily manipulated, and moreover, thousands of embryos can be obtained in a single day.  Indeed, ''Xenopus'' was the first vertebrate animal for which methods were developed to allow rapid analysis of gene function using misexpression (by mRNA injection<ref>{{cite journal | vauthors = Gurdon JB, Lane CD, Woodland HR, Marbaix G | title = Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells | journal = Nature | volume = 233 | issue = 5316 | pages = 177–182 | date = September 1971 | pmid = 4939175 | doi = 10.1038/233177a0 | s2cid = 4160808 | bibcode = 1971Natur.233..177G }}</ref>).  Injection of mRNA in ''Xenopus'' that led to the cloning of interferon.<ref>{{cite journal | vauthors = Reynolds FH, Premkumar E, Pitha PM | title = Interferon activity produced by translation of human interferon messenger RNA in cell-free ribosomal systems and in Xenopus oöcytes | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 72 | issue = 12 | pages = 4881–4885 | date = December 1975 | pmid = 1061077 | pmc = 388836 | doi = 10.1073/pnas.72.12.4881 | doi-access = free | bibcode = 1975PNAS...72.4881R }}</ref>  Moreover, the use of morpholino-antisense oligonucleotides for gene knockdowns in vertebrate embryos, which is now widely used, was first developed by Janet Heasman using ''Xenopus''.<ref>{{cite journal | vauthors = Heasman J, Kofron M, Wylie C | title = Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach | journal = Developmental Biology | volume = 222 | issue = 1 | pages = 124–134 | date = June 2000 | pmid = 10885751 | doi = 10.1006/dbio.2000.9720 | doi-access = free }}</ref>

In recent years, these approaches have played in important role in studies of human disease genes.  The mechanism of action for several genes mutated in human cystic kidney disorders (e.g. [[nephronophthisis]]) have been extensively studied in ''Xenopus'' embryos, shedding new light on the link between these disorders, [[ciliogenesis]] and [[Wnt signaling]].<ref>{{cite journal | vauthors = Schäfer T, Pütz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A, Walz G | display-authors = 6 | title = Genetic and physical interaction between the NPHP5 and NPHP6 gene products | journal = Human Molecular Genetics | volume = 17 | issue = 23 | pages = 3655–3662 | date = December 2008 | pmid = 18723859 | pmc = 2802281 | doi = 10.1093/hmg/ddn260 }}</ref>  ''Xenopus'' embryos have also provided a rapid test bed for validating newly discovered disease genes.  For example, studies in ''Xenopus'' confirmed and elucidated the role of ''PYCR1'' in [[cutis laxa]] with progeroid features.<ref>{{cite journal | vauthors = Reversade B, Escande-Beillard N, Dimopoulou A, Fischer B, Chng SC, Li Y, Shboul M, Tham PY, Kayserili H, Al-Gazali L, Shahwan M, Brancati F, Lee H, O'Connor BD, Schmidt-von Kegler M, Merriman B, Nelson SF, Masri A, Alkazaleh F, Guerra D, Ferrari P, Nanda A, Rajab A, Markie D, Gray M, Nelson J, Grix A, Sommer A, Savarirayan R, Janecke AR, Steichen E, Sillence D, Hausser I, Budde B, Nürnberg G, Nürnberg P, Seemann P, Kunkel D, Zambruno G, Dallapiccola B, Schuelke M, Robertson S, Hamamy H, Wollnik B, Van Maldergem L, Mundlos S, Kornak U | display-authors = 6 | title = Mutations in PYCR1 cause cutis laxa with progeroid features | journal = Nature Genetics | volume = 41 | issue = 9 | pages = 1016–1021 | date = September 2009 | pmid = 19648921 | doi = 10.1038/ng.413 | s2cid = 10221927 }}</ref>

Transgenic ''Xenopus'' for studying transcriptional regulation of human disease genes:  ''Xenopus'' embryos develop rapidly, so transgenesis in ''Xenopus'' is a rapid and effective method for analyzing genomic regulatory sequences.  In a recent study, mutations in the ''SMAD7'' locus were revealed to associate with human [[colorectal cancer]].  The mutations lay in conserved, but noncoding sequences, suggesting these mutations impacted the patterns of ''SMAD7'' transcription.  To test this hypothesis, the authors used ''Xenopus'' transgenesis, and revealed this genomic region drove expression of [[Green fluorescent protein|GFP]] in the hindgut.  Moreover, transgenics made with the mutant version of this region displayed substantially less expression in the hindgut.<ref>{{cite journal | vauthors = Pittman AM, Naranjo S, Webb E, Broderick P, Lips EH, van Wezel T, Morreau H, Sullivan K, Fielding S, Twiss P, Vijayakrishnan J, Casares F, Qureshi M, Gómez-Skarmeta JL, Houlston RS | display-authors = 6 | title = The colorectal cancer risk at 18q21 is caused by a novel variant altering SMAD7 expression | journal = Genome Research | volume = 19 | issue = 6 | pages = 987–993 | date = June 2009 | pmid = 19395656 | pmc = 2694486 | doi = 10.1101/gr.092668.109 }}</ref>

''Xenopus'' cell-free extracts for biochemical studies of proteins encoded by human disease genes: A unique advantage of the ''Xenopus'' system is that cytosolic extracts contain both soluble cytoplasmic and nuclear proteins (including chromatin proteins). This is in contrast to cellular extracts prepared from somatic cells with already distinct cellular compartments.  ''Xenopus'' egg extracts have provided numerous insights into the basic biology of cells with particular impact on cell division and the DNA transactions associated with it (see below).

Studies in ''Xenopus'' egg extracts have also yielded critical insights into the mechanism of action of human disease genes associated with genetic instability and elevated cancer risk, such as ataxia telangiectasia, ''[[BRCA1]]'' inherited breast and ovarian cancer, ''Nbs1'' Nijmegen breakage syndrome, ''RecQL4'' Rothmund-Thomson syndrome, ''[[c-Myc]]'' oncogene and FANC proteins ([[Fanconi anemia]]).<ref>{{cite journal | vauthors = Joukov V, Groen AC, Prokhorova T, Gerson R, White E, Rodriguez A, Walter JC, Livingston DM | display-authors = 6 | title = The BRCA1/BARD1 heterodimer modulates ran-dependent mitotic spindle assembly | journal = Cell | volume = 127 | issue = 3 | pages = 539–552 | date = November 2006 | pmid = 17081976 | doi = 10.1016/j.cell.2006.08.053 | s2cid = 17769149 | doi-access = free }}</ref><ref>{{cite journal | vauthors = You Z, Bailis JM, Johnson SA, Dilworth SM, Hunter T | title = Rapid activation of ATM on DNA flanking double-strand breaks | journal = Nature Cell Biology | volume = 9 | issue = 11 | pages = 1311–1318 | date = November 2007 | pmid = 17952060 | doi = 10.1038/ncb1651 | s2cid = 17389213 }}</ref><ref name="Checkpoint signaling from a single">{{cite journal | vauthors = Ben-Yehoyada M, Wang LC, Kozekov ID, Rizzo CJ, Gottesman ME, Gautier J | title = Checkpoint signaling from a single DNA interstrand crosslink | journal = Molecular Cell | volume = 35 | issue = 5 | pages = 704–715 | date = September 2009 | pmid = 19748363 | pmc = 2756577 | doi = 10.1016/j.molcel.2009.08.014 }}</ref><ref>{{cite journal | vauthors = Sobeck A, Stone S, Landais I, de Graaf B, Hoatlin ME | title = The Fanconi anemia protein FANCM is controlled by FANCD2 and the ATR/ATM pathways | journal = The Journal of Biological Chemistry | volume = 284 | issue = 38 | pages = 25560–25568 | date = September 2009 | pmid = 19633289 | pmc = 2757957 | doi = 10.1074/jbc.M109.007690 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Dominguez-Sola D, Ying CY, Grandori C, Ruggiero L, Chen B, Li M, Galloway DA, Gu W, Gautier J, Dalla-Favera R | display-authors = 6 | title = Non-transcriptional control of DNA replication by c-Myc | journal = Nature | volume = 448 | issue = 7152 | pages = 445–451 | date = July 2007 | pmid = 17597761 | doi = 10.1038/nature05953 | s2cid = 4422771 | bibcode = 2007Natur.448..445D }}</ref>

''Xenopus'' oocytes for studies of gene expression and channel activity related to human disease:  Yet another strength of ''Xenopus'' is the ability to rapidly and easily assay the activity of channel and transporter proteins using expression in oocytes.  This application has also led to important insights into human disease, including studies related to [[Trypanosomatid|trypanosome]] transmission,<ref>{{cite journal | vauthors = Dean S, Marchetti R, Kirk K, Matthews KR | title = A surface transporter family conveys the trypanosome differentiation signal | journal = Nature | volume = 459 | issue = 7244 | pages = 213–217 | date = May 2009 | pmid = 19444208 | pmc = 2685892 | doi = 10.1038/nature07997 | bibcode = 2009Natur.459..213D }}</ref> [[Epilepsy]] with [[ataxia]] and [[sensorineural deafness]]<ref>{{cite journal | vauthors = Bockenhauer D, Feather S, Stanescu HC, Bandulik S, Zdebik AA, Reichold M, Tobin J, Lieberer E, Sterner C, Landoure G, Arora R, Sirimanna T, Thompson D, Cross JH, van't Hoff W, Al Masri O, Tullus K, Yeung S, Anikster Y, Klootwijk E, Hubank M, Dillon MJ, Heitzmann D, Arcos-Burgos M, Knepper MA, Dobbie A, Gahl WA, Warth R, Sheridan E, Kleta R | display-authors = 6 | title = Epilepsy, ataxia, sensorineural deafness, tubulopathy, and KCNJ10 mutations | journal = The New England Journal of Medicine | volume = 360 | issue = 19 | pages = 1960–1970 | date = May 2009 | pmid = 19420365 | pmc = 3398803 | doi = 10.1056/NEJMoa0810276 }}</ref>  Catastrophic [[cardiac arrhythmia]] ([[Long-QT syndrome]])<ref>{{cite journal | vauthors = Gustina AS, Trudeau MC | title = A recombinant N-terminal domain fully restores deactivation gating in N-truncated and long QT syndrome mutant hERG potassium channels | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 106 | issue = 31 | pages = 13082–13087 | date = August 2009 | pmid = 19651618 | pmc = 2722319 | doi = 10.1073/pnas.0900180106 | doi-access = free | bibcode = 2009PNAS..10613082G }}</ref>  and Megalencephalic leukoencephalopathy.<ref>{{cite journal | vauthors = Duarri A, Teijido O, López-Hernández T, Scheper GC, Barriere H, Boor I, Aguado F, Zorzano A, Palacín M, Martínez A, Lukacs GL, van der Knaap MS, Nunes V, Estévez R | display-authors = 6 | title = Molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts: mutations in MLC1 cause folding defects | journal = Human Molecular Genetics | volume = 17 | issue = 23 | pages = 3728–3739 | date = December 2008 | pmid = 18757878 | pmc = 2581428 | doi = 10.1093/hmg/ddn269 }}</ref>

Gene editing by the CRISPR/CAS system has recently been demonstrated in ''Xenopus'' ''tropicalis''<ref>{{cite journal | vauthors = Blitz IL, Biesinger J, Xie X, Cho KW | title = Biallelic genome modification in F(0) Xenopus tropicalis embryos using the CRISPR/Cas system | journal = Genesis | volume = 51 | issue = 12 | pages = 827–834 | date = December 2013 | pmid = 24123579 | pmc = 4039559 | doi = 10.1002/dvg.22719 }}</ref><ref>{{cite journal | vauthors = Nakayama T, Fish MB, Fisher M, Oomen-Hajagos J, Thomsen GH, Grainger RM | title = Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis | journal = Genesis | volume = 51 | issue = 12 | pages = 835–843 | date = December 2013 | pmid = 24123613 | pmc = 3947545 | doi = 10.1002/dvg.22720 }}</ref> and ''Xenopus laevis''.<ref>{{cite journal | vauthors = Wang F, Shi Z, Cui Y, Guo X, Shi YB, Chen Y | title = Targeted gene disruption in Xenopus laevis using CRISPR/Cas9 | language = En | journal = Cell & Bioscience | volume = 5 | issue = 1 | pages = 15 | date = 2015-04-14 | pmid = 25897376 | pmc = 4403895 | doi = 10.1186/s13578-015-0006-1 | doi-access = free }}</ref> This technique is being used to screen the effects of human disease genes in ''Xenopus'' and the system is sufficiently efficient to study the effects within the same embryos that have been manipulated.<ref>{{cite journal | vauthors = Bhattacharya D, Marfo CA, Li D, Lane M, Khokha MK | title = CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus | journal = Developmental Biology | volume = 408 | issue = 2 | pages = 196–204 | date = December 2015 | pmid = 26546975 | pmc = 4684459 | doi = 10.1016/j.ydbio.2015.11.003 | series = Modeling Human Development and Disease in Xenopus }}</ref>