Mutation in mice may have affected research results

Lab mouse

Researchers say they have discovered a mutation in a subline of C57BL/6 mice that could compromise results from previous studies.

“We found an unexpected mutation with potentially important consequences in strains of mice that had been separately engineered in labs in California and Japan,” said Shiv Pillai, MD, PhD, of The Ragon Institute of MGH, MIT and Harvard in Cambridge, Massachusetts.

“[W]e traced the problem to a subline of B6 mice from one specific company that have been sold in Asia, North America, Europe, and Israel. We have notified this company—Harlan Laboratories, which is now part of Envigo—of our findings, and they have been very responsive and will use approaches we have provided to check all of their colonies.”

Dr Pillai and his colleagues described their discovery of the mutation in Cell Reports.

The team noted that, in immunological studies, mutant mice are backcrossed for many generations into B6 mice so that all genes other than the mutant gene are derived from the B6 strain. This allows the comparison of data from laboratories in different parts of the world and simplifies creating mice with mutations in several genes.

A 2009 study out of Dr Pillai’s lab showed that 2 strains of mice engineered to lack the Siae or Cmah genes—both of which code for enzymes involved with sialic acid proteins—also had significant defects in the development of B cells, which were assumed to be the result of the knockout genes.

However, when Siae-deficient mice were further backcrossed with a different group of C57BL/6 mice, the result was a strain of Siae-knockout mice that did not have the B-cell development defects. A newly engineered strain of mice with a different Siae mutation also had normal B-cell development.

Detailed genetic sequencing of the first knockout line revealed a previously unsuspected mutation in a gene called Dock2, located on a chromosome 11, instead of chromosome 9 where Siae is located.

The same mutation was previously reported in 2 colonies of a different knockout mouse developed in Japan.

Dr Pillai’s team also found the Dock2 mutation in a completely different group of mice from the University of California, San Diego—where their Siae-mutant strain had been developed—and realized that 3 different engineered strains with the same unwanted mutation had probably acquired it from a common source. The team eventually traced it back to a subline of C57BL/6 mice from Harlan/Envigo.

Since most research papers using C57BL/6 mice or other such “background” strains do not indicate the specific subline, the researchers said they have no way of knowing how many studies might be affected by their findings.

But they hope the publication of these results will alert other research teams to the potential need to review their results.

“While embryonic stem cells from C57BL/6 mice have recently become available, which allows the generation of knockout strains with less backcrossing, B6 mice are used for many different kinds of experiments, including as controls,” Dr Pillai said.

“Researchers who have used them need to re-genotype the mice to look for the Dock2 mutation and, if they find it, check to see whether their results are preserved if the Dock2 mutation is bred out.”

Lab mouse

Researchers say they have discovered a mutation in a subline of C57BL/6 mice that could compromise results from previous studies.

“We found an unexpected mutation with potentially important consequences in strains of mice that had been separately engineered in labs in California and Japan,” said Shiv Pillai, MD, PhD, of The Ragon Institute of MGH, MIT and Harvard in Cambridge, Massachusetts.

“[W]e traced the problem to a subline of B6 mice from one specific company that have been sold in Asia, North America, Europe, and Israel. We have notified this company—Harlan Laboratories, which is now part of Envigo—of our findings, and they have been very responsive and will use approaches we have provided to check all of their colonies.”

Dr Pillai and his colleagues described their discovery of the mutation in Cell Reports.

The team noted that, in immunological studies, mutant mice are backcrossed for many generations into B6 mice so that all genes other than the mutant gene are derived from the B6 strain. This allows the comparison of data from laboratories in different parts of the world and simplifies creating mice with mutations in several genes.

A 2009 study out of Dr Pillai’s lab showed that 2 strains of mice engineered to lack the Siae or Cmah genes—both of which code for enzymes involved with sialic acid proteins—also had significant defects in the development of B cells, which were assumed to be the result of the knockout genes.

However, when Siae-deficient mice were further backcrossed with a different group of C57BL/6 mice, the result was a strain of Siae-knockout mice that did not have the B-cell development defects. A newly engineered strain of mice with a different Siae mutation also had normal B-cell development.

Detailed genetic sequencing of the first knockout line revealed a previously unsuspected mutation in a gene called Dock2, located on a chromosome 11, instead of chromosome 9 where Siae is located.

The same mutation was previously reported in 2 colonies of a different knockout mouse developed in Japan.

Dr Pillai’s team also found the Dock2 mutation in a completely different group of mice from the University of California, San Diego—where their Siae-mutant strain had been developed—and realized that 3 different engineered strains with the same unwanted mutation had probably acquired it from a common source. The team eventually traced it back to a subline of C57BL/6 mice from Harlan/Envigo.

Since most research papers using C57BL/6 mice or other such “background” strains do not indicate the specific subline, the researchers said they have no way of knowing how many studies might be affected by their findings.

But they hope the publication of these results will alert other research teams to the potential need to review their results.

“While embryonic stem cells from C57BL/6 mice have recently become available, which allows the generation of knockout strains with less backcrossing, B6 mice are used for many different kinds of experiments, including as controls,” Dr Pillai said.

“Researchers who have used them need to re-genotype the mice to look for the Dock2 mutation and, if they find it, check to see whether their results are preserved if the Dock2 mutation is bred out.”

Lab mouse

Researchers say they have discovered a mutation in a subline of C57BL/6 mice that could compromise results from previous studies.

“We found an unexpected mutation with potentially important consequences in strains of mice that had been separately engineered in labs in California and Japan,” said Shiv Pillai, MD, PhD, of The Ragon Institute of MGH, MIT and Harvard in Cambridge, Massachusetts.

“[W]e traced the problem to a subline of B6 mice from one specific company that have been sold in Asia, North America, Europe, and Israel. We have notified this company—Harlan Laboratories, which is now part of Envigo—of our findings, and they have been very responsive and will use approaches we have provided to check all of their colonies.”

Dr Pillai and his colleagues described their discovery of the mutation in Cell Reports.

The team noted that, in immunological studies, mutant mice are backcrossed for many generations into B6 mice so that all genes other than the mutant gene are derived from the B6 strain. This allows the comparison of data from laboratories in different parts of the world and simplifies creating mice with mutations in several genes.

A 2009 study out of Dr Pillai’s lab showed that 2 strains of mice engineered to lack the Siae or Cmah genes—both of which code for enzymes involved with sialic acid proteins—also had significant defects in the development of B cells, which were assumed to be the result of the knockout genes.

However, when Siae-deficient mice were further backcrossed with a different group of C57BL/6 mice, the result was a strain of Siae-knockout mice that did not have the B-cell development defects. A newly engineered strain of mice with a different Siae mutation also had normal B-cell development.

Detailed genetic sequencing of the first knockout line revealed a previously unsuspected mutation in a gene called Dock2, located on a chromosome 11, instead of chromosome 9 where Siae is located.

The same mutation was previously reported in 2 colonies of a different knockout mouse developed in Japan.

Dr Pillai’s team also found the Dock2 mutation in a completely different group of mice from the University of California, San Diego—where their Siae-mutant strain had been developed—and realized that 3 different engineered strains with the same unwanted mutation had probably acquired it from a common source. The team eventually traced it back to a subline of C57BL/6 mice from Harlan/Envigo.

Since most research papers using C57BL/6 mice or other such “background” strains do not indicate the specific subline, the researchers said they have no way of knowing how many studies might be affected by their findings.

But they hope the publication of these results will alert other research teams to the potential need to review their results.

“While embryonic stem cells from C57BL/6 mice have recently become available, which allows the generation of knockout strains with less backcrossing, B6 mice are used for many different kinds of experiments, including as controls,” Dr Pillai said.

“Researchers who have used them need to re-genotype the mice to look for the Dock2 mutation and, if they find it, check to see whether their results are preserved if the Dock2 mutation is bred out.”