Читать книгу Pathology of Genetically Engineered and Other Mutant Mice - Группа авторов - Страница 68

Targeted mutations were initially generated to produce mutant mice with inactivation of one specific gene, the so‐called “knockout” mouse lines. Targeted mutations can also have a foreign gene or gene segment inserted into a target gene, resulting in expression of the foreign gene under control of the endogenous promoter. These are termed “knock‐in” mutations. In these cases, the inserted gene symbol is included in parentheses as part of the targeted allele symbol. However, reporter symbols, such as GFP, are not included in allele symbols. Details describing the specifics of knock‐in constructs should be provided in databases or publications, and not in the nomenclature.

In the targeted knock‐in mutation En1^{tm1(Otx2)Wrst}, the coding region of the engrailed 1 gene (En1) was replaced by the orthodenticle homeobox 2 (Otx2) gene, originating from the W. Wurst laboratory [45]. For the Cd19 antigen targeted knock‐in mutation Cd19^tm1(cre)Cgn, cre‐recombinase was inserted in‐frame in exon 1 [46]. The allele expresses cre‐recombinase specifically in B‐lineage cells throughout development. The apolipoprotein E targeted knock‐in mutation (Apoe^{tm1(APOE*2)Mae}) has a DNA fragment containing exons 2–4 of a human APOE2 isoform replacing the equivalent portion of the mouse Apoe gene. The human protein is expressed from this allele and the endogenous mouse protein is undetectable.

When a targeting vector is used to generate multiple germline‐transmissible alleles, such as in the cre‐Lox system, the original knock‐in of loxP would follow the regular tm designation rules. If a second heritable allele was then generated after mating with a cre‐recombinase transgenic mouse, it would retain the parental designation followed by a decimal point and serial number. For example, Tfam^tm1Lrsn designates a targeted mutation where loxP was inserted into the transcription factor A, mitochondrial (Tfam) gene [47]. Tfam^tm1.1Lrsn (note: tm1Lrsn vs. tm1.1Lrsn) designates the derivative germline transmissible allele generated after mating with a cre‐recombinase transgenic mouse. Note that somatic events generated in offspring from a Tfam^tm1Lrsn bearing mouse and a cre‐recombinase transgenic that cause disruption of Tfam in selective tissues would not be part of strain nomenclature.

Mutagenesis using targeted endonuclease‐cleavage technologies (CRISPR, Zinc Finger endonuclease, or TALEN) create site‐targeted genomic changes by either non‐homologous end joining resulting in random indels (insertion/deletions), or homologous recombination with a donor plasmid to generate specific sequence alterations (floxed, point mutations, insertion of exogenous sequence, etc.). While there is no standard for naming endonuclease‐mediated mutations across species, mouse, rat, and Xenopus designate such mutation with “em” [48] followed with the serial number of endonuclease‐mediated mutation for the generating laboratory, and the ILAR‐registered laboratory code (e.g. Abcb1a^em1Sbkv for endonuclease‐mediated mutation 1 from the laboratory of Dr. Sergei B Koralov in adenosine triphosphate (ATP)‐binding cassette, sub‐family B [MDR/TAP], member 1A gene) [49]. Endonuclease‐mediated mutation nomenclature mirrors targeted allele nomenclature in regard to inserted expressed sequences (http://www.informatics.jax.org/mgihome/nomen/gene.shtml#endim). When endonuclease‐mediated mutations are used to produce multigenic mutations either through direct targeting or recombination between previously targeted sites, chromosomal aberration nomenclature is used in the absence of a marker designating the region affected (to be added to the nomen webpage). For example, Del(5Kit‐Nmu)2Staka (deletion, Chr 5, Satoru Takahashi 2) describes the 1.07 Mb deletion produced by CRISPR/Cas targeting of a region 240 kb upstream of Kit and 81 kb downstream of Nmu [50].

Gene trap alleles are produced by random integration of constructs containing splice acceptors and other sequences that prematurely terminated transcription disrupting gene expression [51]. Alleles generated by the integration of a gene trap are represented with “Gt,” the mutant cell line in parenthesis, and ILAR‐registered laboratory code. For example, in Dnajb9^{Gt(KST256)Byg} (gene trap KST256 from BayGenomics in DnaJ heat shock protein family [Hsp40] member B9), a gene trap with a splice donor and β‐galactosidase gene is inserted into intron producing a hypomorphic allele [52].

Enhancer traps are specialized transgenes. One utility of these transgenes is in creating cre‐recombinase driver lines. Enhancer traps of this type that are currently being created may include a minimal promoter, introns, a cre‐recombinase cassette (sometimes fused with another element such as ERT2), and polyA sites from different sources. Nomenclature for these enhancer traps consists of 4 parts as follows: Et, prefix for enhancer trap; cre recombinase cassette, portion in parentheses (for example, cre, icre, or cre/ERT2 [if fused with ERT2]); line number or serial number to designate lab trap number or serial number; Lab code: ILAR code identifying the creator of this enhancer trap; Examples include: Et(icre)754Rdav (for Enhancer trap 754, Ron Davis) and Et(cre/ERT2)2047Rdav (for Enhancer trap 2047, Ron Davis) [53].

Transposon‐induced mutations utilize transposases (e.g. sleeping beauty) to mobilize integrated transposon concatemers flanked by long terminal repeats (LTR) to insert into small base pair recognition sequences [54]. When integration of the transposon does not disrupt the LTR, these elements can be remobilized and “hop” into different sites located either on the same chromosome or a different one. Transposons can insert sequences to be expressed as well as disrupting gene expression at integration site. Transposon‐induced alleles are denoted with “Tn,” the transposon concatemer (specifying the transposase), a line number (followed by a period “.” and additional serial number in the case of remobilization), and ILAR‐registered laboratory code. In the transposon‐induced mutation Slc16a10^{Tn(sb‐lacZ,GFP)1.4Tcb} (line 4 containing reintegration of a sleeping beauty‐specific transposon that expressed lacZ and GFP from Dr. Thomas Brodnicki), transposition of the expression cassette into intron 1 produces a null allele [55].

In many cases, a large number of transgenic lines are made from the same gene construct and only differ by tissue specificity of expression. The most common of these are transgenes that use reporter constructs or recombinases (e.g. GFP, lacZ, cre), where the promoter should be specified as the first part of the gene insertion designation, separated by a hyphen from the reporter or recombinase designation. The SV40 large T antigen is another example. The use of promoter designations is helpful in such cases. Examples include Tg(Wnt1‐LacZ)206Amc, in which the mouse has a LacZ transgene with a wingless‐type mouse mammary tumor virus (MMTV) integration site family, member 1 (Wnt1) promoter, from mouse line 206 created in the laboratory of Andrew McMahon [56]. Another example is Tg(Zp3‐cre)3Mrt in which the cre transgene has a zona pellucida glycoprotein 3 (Zp3) promoter, the third transgenic mouse line from the laboratory of Gail Martin [57].