If he cannot generate a mutant, it is most likely because the mutant phenotype is lethal. In theory, this researcher was successful in mutating the target gene, however the result of the mutation was a failure during development. This would prevent any potential offspring with the mutation from developing, resulting in no viable specimens for the study.

Many proteins are conserved throughout evolution, but can still be easily mutated. For example, the gene for the protein hemoglobin is well-preserved between animal species, but can still be manipulated to generate animal models for various disorders. The distinction between heterochromatin and euchromatin doesn't really explain why the researcher would be unable to generate a mutant.

All of the given answers could potentially be used in a mutagenesis procedure.

For PCR, special primers can be designed to introduce site-directed mutagenesis. UV light is a common mutagen and could be used to make random mutations throughout the genome. Homologous recombination can be used to incorporate a non-functional copy of a gene into an organism.

A chimeric mouse results from implanting altered stem cells into a fertile mother. Her offspring will consist of mice made partially of her own stem cells and partially from the stem cells that were implanted by the researcher. The term chimera does not necessarily reflect upon the genotype of the mice.

CRISPR/Cas9 utilizes a gRNA sequence that directs the endonuclease activity of Cas9 to specific PAM recognition sites within the genome. TALENs harbor a DNA binding domain and a nuclease domain. Binding of the TALEN to a specific loci activates the nuclease domain to cut at that specific locus. ZFNs, similar to TALENs, have a Zinc-finger domain that binds specific DNA sequences and a DNA-cleavage domain. The above technologies are programmable to target varying genomic loci. Restriction endonucleases alone, however, are not utilized to stably alter genomes.

The correct answer is all of the answers. Chromatin immunoprecipitation with sequencing uses an antibody specific to the transcription factor to pull down crosslinked transcription factor-DNA complexes. Sequencing of this DNA then determines the promoters of genes that this transcription factor binds. Chromatin immunoprecipitation with microarray analysis is similar, however, the DNA bound in complex is analyzed by a designed microarray of select genes (promoters). Morpholinos are anti-sense DNA oligomers that prevent translation of their target genes, and as such, are an efficient way to knockdown gene expression. By knocking down a transcription factor, we can measure differentially regulated genes compared to a control. The read-out experiment for measuring up or down-regulated genes in the knockout condition can be either by RNA-sequencing or by microarray analysis.

Morpholinos, short hairpin RNAs, and small interfering RNAs are methodologies to transiently knockdown the expression of a specific gene by complementary base pairing with the transcribed mRNA of gene of interest, inhibiting translation. CRISPR/Cas9 permanently mutates a gene of interest by introducing a double-stranded DNA break within the gene. All of these technologies are widely used in disruption of gene expression.

正确的答案是protospacer相邻的图案(PAMs). gRNAs guide Cas9 to a specific genomic loci, however, in order for Cas9 to introduce a DNA break, a PAM, which are 5' NGG 3' sequences, must be present at the loci as well. TATA boxes are functional elements in eukaryotic promoters that promote activation of transcription. Trans-activating CRISPR-RNAs are important for pathogen defense in bacteria and archaea, but are not utilized in the gene editing technology.