Table 1 In vivo phenotypic studies of lncRNAs

H19

Replacement of a 3-kb gene region and 10 kb of 5′ flanking sequence of the lncRNA with a neomycin resistance cassette

Overgrowth in the animals inheriting the H19 mutation from their mothers compared to those inheriting it from their fathers

N

Y⁴

HR

[32]

H19

Replacement of the entire lncRNA transcription unit with a neo cassette

Overgrowth (8%)

N

Y⁵

HR

[33]

H19

Same as above

Overgrowth in the lncRNA knockouts reflected in general (up to 20%) increase in weight. Corresponding decrease in weight was observed in knockout animals overexpressing the lncRNA.

Y

HR

[34]

H19

Same as in Ripoche et al. [33]. The E6.5 embryos were grafted into the wild-type or Igf2^−/− recipient mice to induce teratocarcinomas.

Increased weight of experimental teratocarcinomas

N

HR

[35]

Knockout animals from Ripoche et al. [33] were bred with Apc^∆14/+ mice

Increased number of adenomas compared with their Apc littermates

N

HR

Maternal heterozygotes of the H19 knockout mice same as in Leighton et al. [32] were bred with CRP-Tag 60-3 male mice.

Acceleration of liver tumor development

N

HR

H19

Same as in Ripoche et al. [33]

Muscle hypertrophy and hyperplasia. A 50% reduction in the number of satellite cells

Y

HR

[36]

H19

Same as in Ripoche et al. [33]

Increased tumor development after carcinogen diethylnitrosamine treatment

N

HR

[37]

H19

Same as in Ripoche et al. [33]. The H19 heterozygous male knockout mice were bred with the wild-type mice to generate paternal and maternal knockouts.

Increased liver weights immediately after birth

N

HR

[38]

roX1/2⁶

Deletion of the roX2 gene, transposon insertion inactivation, or partial deletion of the roX1 gene

Male-specific reduction in viability in the animals lacking both roX1 and roX2 genes

Y

[39]

Xist

Replacement of most of the lncRNA transcription unit with a neo cassette while leaving the promoter intact

Females carrying the Xist knockout on the paternal chromosome exhibited severe growth retardation and early embryonic lethality.

N

HR

[40]

Xist

Inversion of the exon 1 and deletion of the exon 4

Embryonic lethality in paternal knockout mice

N

HR and Cre

[41]

Xist

Mice with loxP sites inserted into Xist intron 3 and 5 kb upstream of the somatic cell promoter (Xist^2lox/2lox) were bred with Vav.Cre mice to conditionally delete Xist in murine hematopoietic stem cells.

Females developed a highly aggressive myeloproliferative neoplasm and myelodysplastic syndrome with 100% penetrance.

N

Cre

[42]

Xist

The Xist^fl/fl or Xist^∆/fl mice generated using the same knockout strategy as above were crossed with Sox2-Cre mice to conditionally delete Xist in the epiblast lineage.

Females exhibited retarded growth, abnormal development of some organs, and failure to survive past weaning age.

N

Cre

[43]

Xist

Xist^lox/lox mice generated using the same knockout strategy as above were crossed with MMTV-Cre mice to generate animals with a mammary-specific knockout of Xist.

Acceleration of primary tumor growth in mammary glands and metastases in the brain

N

Cre

[44]

Malat1

Gene inactivation using insertion of the lacZ gene and polyadenylation signals immediately downstream of the transcriptional start site

No apparent phenotype

N/A

HR

[25]

Malat1

Same as above, but bred to MMTV-PyMT mice to generate MMTVPyMT;Malat1^−/− females

Promotion of lung metastasis in the knockout animals with breast cancer, contradictory to the results of Arun et al. (2016) [47]

Y

HR

[45]

Malat1

Deletion of a ~ 3-kb genomic region containing the 5′ end of the Malat1 gene and its promoter

No apparent phenotype

N/A

HR, FLP, and Cre

[46]

Malat1

Same as Zhang et al. [46], but bred with MMTV-PyMT male mice

Reduction of branching morphogenesis in the MMTV-PyMT and Her2/neu-amplified tumor organoids, increase of cell adhesion, and loss of migration

N

HR

[47]

Malat1

Same as Zhang et al. [46]

Increased brain infarct size, worsened neurological scores, and reduced sensorimotor functions

N

HR

[48]

Malat1

Deletion of the complete 6982 bp Malat1 transcript sequence plus 251 bp upstream of the Malat1 transcription start site and 322 bp downstream of the Malat1 transcript end

No apparent phenotype

N/A

HR and Cre

[23]

Malat1

Same as above

No apparent phenotype

N/A

HR and Cre

[24]

Malat1

Same as in Eissmann et al. [23], but crossed with Apoe^−/− mice

After a high-fat diet,the Apoe^−/−Malat1^−/− mice showed increased plaque size and infiltration of inflammatory CD45+ cells, as well as enhanced adhesion of myeloid cells to atherosclerotic arteries compared to the Apoe^−/−Malat1^+/+ mice.

N

HR and Cre

[49]

Hotair

Deletion of the exons 1 and 2

Three notable anatomical phenotypes related to skeleton malformations

N

HR and Cre

[50]

Hotair

The same knockout strain as above, however, crossed into a different genetic background

No apparent phenotype attributable to the lncRNA, failure to reproduce the phenotypes above

N/A

HR and Cre

[26]

Hotair

Replacement of a 2.3-kb genomic sequence from exon 1 to the last annotated exon with a lacZ-neomycin resistance cassette

Morphological malformations in caudal vertebra

N

HR

[51]

Neat1

Gene inactivation using insertion of the lacZ gene and polyadenylation signals immediately downstream of the transcriptional start site

No apparent phenotypes except for disappearance of paraspeckles

N/A

HR

[52]

Neat1

Same as above

Stochastic failure to become pregnant in a subpopulation of the knockout animals

N

HR

[53]

Neat1

Presumably the same as in Nakagawa et al. [52]

Aberrant mammary gland morphogenesis and lactation defects

N

[54]

Neat1

Same as in Nakagawa et al. [52]

Preneoplastic cells were sensitized to DNA-damage-induced cell death, and skin tumorigenesis was impaired.

N

HR

[55]

Neat1

Same as in Nakagawa et al. [52]

Decrease of neointima formation following vascular injury

N

HR

[56]

Neat1

Deletion of the entire lncRNA transcription unit

Reduction of inflammatory responses

N

Cas9

[57]

Fendrr

Replacement of exon 1 with a transcriptional stop signal (3x pA)

Embryonic lethality and impairment of the heart and body wall

Y

HR

[58]

Firre

Deletion of the entire Firre gene body and promoter

Cell-specific hematopoietic phenotypes

Y

HR and Cre

[59]

lncKdm2b

Deletion of a 838-bp fragment containing the exon 2

Impaired embryonic stem cell self-renewal and early embryonic lethality

Y

Cas9

[60]

PCFL

Deletion of a 6475-bp region containing PCFL and adjacent sequences

Improved heart function and reduced cardiac fibrosis after myocardial infarction in heterozygous knockout animals

Y

Cas9

[61]

Chaer

Deletion of exon 2

Attenuated cardiac hypertrophy and blunted pathological fibrosis following trans-aortic constriction

N (in vitro rescue only)

Cas9

[62]

linc1405

Deletion of exon 2

Impaired heart development and function

N (in vitro rescue only)

Cas9

[63]

lincRNA-EPS

Replacement of the entire 4-kb genomic locus with a neomycin cassette

Enhanced inflammation and lethality following endotoxin challenge

N (in vitro rescue only)

HR

[64]

lncKdm2b

Same as in Ye et al. [60]

Early embryonic lethality. Impaired intestinal group 3 innate lymphoid cell (ILC3) maintenance and proliferation

N (in vitro rescue only)

Cas9

[65]

Mice with loxP elements flanking the exon 2 of lncKdm2b were crossed with Vav-Cre⁺ mice to generate animals with a conditional deletion of lncKdm2b from the bone marrow.

Markedly decreased absolute numbers of ILC3s

N

Cas9 and Cre

Mice with loxP elements flanking the exon 2 of lncKdm2b were crossed with Rorc-Cre⁺mice to generate mice with conditional deletion of lncKdm2b from ILC3s.

Remarkably decreased numbers of all ILC3 subpopulations

N

Cas9 and Cre

ANRIL

Deletion of the 70-kb region on Chr 4 containing the mouse gene aligning to human 58-kb non-coding CAD risk interval

Showed a protective effect on diabetic mouse kidneys (lowering of urine volume and urine albumin levels in comparison with the wild-type diabetic animals)

N

HR and Cre

[66]

Blnc1

Adipose tissue-specific deletion of the entire gene

Mice with fat-specific inactivation of Blnc1 showed impaired cold-induced thermogenesis and browning and exacerbation of obesity-associated brown fat whitening, adipose tissue inflammation, and fibrosis, leading to a more severe insulin resistance and hepatic steatosis.

N

Cas9 and Cre

[67]

Blnc1

Whole body deletion of the entire gene

Liver X receptor agonist-induced rise in plasma triglyceride and hepatic steatosis was significantly blunted by Blnc1 deficiency.

N

Cas9

[68]

Liver-specific deletion of the entire gene

Abrogation of high-fat diet-induced hepatic steatosis and insulin resistance and prevention of diet-induced nonalcoholic steatohepatitis

Bmncr

Deletion of the 4.92-kb sequence of Bmncr

Decreased bone mass and increased bone marrow adiposity

N

HR

[69]

βlinc1

Replacement of the βlinc1 sequence with the puΔtk-EM7-kan cassette

Defective islet development and glucose-intolerance in the adult mice

N

HR

[70]

Charme

Insertion of a polyadenylation/MAZ cassette into the beginning of the exon 2

Peculiar heart remodeling phenotype (changes in size, structure, and shape of the organ), morphological alteration of skeletal and cardiac muscles, and shortened lifespan

N

Cas9 and HR

[71]

CPR

Replacement of a 2968-bp fragment of CPR gene containing exons 1 and 2 with a neo cassette

Restored heart function after myocardial injury (increased cardiomyocyte proliferation, improved myocardial function, and reduced scar formation)

N

HR

[72]

Cyrano

Deletion of the first half of the exon 3

No overt abnormalities

N/A

HR and Cre

[27]

Dino

Replacement of the bulk of Dino sequence with GFP

Dampened p53 signaling and ameliorated acute radiation syndrome

N

HR

[73]

Inactivation of promoter

Evf2

Insertion of a triple polyadenylation transcription stop site into the exon 1

No apparent phenotype, except for reduced numbers of GABAergic interneurons in early postnatal hippocampus and dentate gyrus

N

HR

[74]

Fendrr

Replacement with a lacZ reporter cassette

Perinatal lethal and lung, heart, and gastrointestinal tract defects

N

HR

[75]

Fendrr

Replacement of a 19-kb genomic sequence from the exon 2 to the last annotated exon with a lacZ-neomycin resistance cassette

Perinatal lethal

N

HR

[51]

Flatr

Deletion of the promoter region and the majority of the exon 1

No reported in vivo phenotype

N

Cas9 and HR

[76]

Deletion of the entirety of the exon 2

Cas9

Flicr

Deletion of the whole exon 2

Flicr-deficient non-obese diabetic female mice showed a significantly reduced rate and incidence of overt diabetes.

N

Cas9

[77]

Gm26878

A 2.3-kb deletion involving the entire lncRNA-encoding gene

Neonatal lethal with low penetrance

N

Cas9

[78]

Gomafu

Deletion of the entire lncRNA gene (157 kb)

Hyperactive behavior with increased sensitivity to the psychostimulant methamphetamine

N

HR and Cre

[79]

Gtl2/Meg3

Replacement of the exons 1–5 (10 kb) with a neo cassette

Maternal knockout pups died within 4 weeks after birth. Paternal knockout mice showed severe growth retardation and perinatal lethality. Homozygous mutants survived and developed into fertile adults.

N

HR

[80]

Gtl2/Meg3

Replacement of the first five exons and adjacent upstream promoter sequences of ~ 300 bp with a neo cassette

Perinatal death and skeletal muscle defects in the mice with the maternal deletion

N

HR

[81]

Gtl2/Meg3

Same as above

Skeletal muscle defects and perinatal death in the maternal knockout animals, as well as increased microvessel formation in the brain

N

HR

[82]

Gtl2/Meg3

Same as above

Increased microvessel formation in the brain

N

HR

[83]

Hottip

Replacement of the 4.8-kb genomic sequence from the exon 1 to the last annotated exon with a lacZ-neomycin resistance cassette

Gastrocnemius muscle defects and hindlimb skeletal malformation

N

HR

[51]

Linc-Brn1b

Replacement with a lacZ reporter cassette

Growth defects (reduced number of intermediate progenitor cells in the cerebral cortex, abnormal cortical lamination and disorganization of the barrel cortex, reduced body weight)

N

HR

[75]

Linc-pint

Replacement with a lacZ reporter cassette

Growth defects (noticeably smaller and reduced body weight)

N

HR

[75]

Linc-pint

Replacement of the 32-kb genomic sequence from the exon 2 to the last annotated exon with a lacZ-neomycin resistance cassette

Growth deficiency (slower growth rate, age-dependent abnormal hindlimb clasping posture, fur loss, lower fat content and femur bone mineral density, decreased muscle mass, and lordokyphosis)

N

HR

[51]

Linc-RAM

Deletion of the exon 2

Delayed muscle regeneration

N

HR and Cre

[84]

lincRNA-p21

Mice with loxP sites flanking the p53 response element in the promoter and exon 1 of the lncRNA were crossed with the Deletor Cre mice to achieve a conditional knockout.

No significant abnormalities

N/A

HR, FLP and Cre

[28]

LncDACH1

LncDACH1^Flox/Flox mice were crossed with α-myosin heavy chain Cre mice to generate mice with a cardiac myocyte-specific knockout of LncDACH1.

Increased calcium transient, cell shortening, and improved cardiac function of transverse aortic constriction induced heart failure mice.

N

Cas9 and Cre

[85]

lncGata6

Deletion of the region from the exon 2 to the exon 4

Impaired stemness of intestinal stem cells (ISCs) and intestinal regeneration

N

Cas9

[86]

Insertion of an SV40 poly(A) (STOP) module into the promoter of the lncRNA

Same as above

Mutation in the lncRNA exon 4

Same as above

Insertion of loxP sequences flanking the exons 2–4 of the lncGata6 locus and establishing Lgr5^GFP-CreERT2; Rosa26^lsl-lacZ; lncGata6^f/f mice

Reduction of ISCs with suppressed cycling and proliferation of ISCs compared to Lgr5^GFP-CreERT2; Rosa26^lsl-lacZ mice

Cas9 and Cre

Lnc-mg

Conditional deletion of the exon 1 in the muscle

Muscle atrophy and the loss of muscular endurance during exercise

N

HR and Cre

[87]

lncOb

Deletion of the 5′ end of the lncRNA first exon

Increased fat mass with reduced plasma leptin levels and lost weight after a leptin treatment

N

Cas9

[88]

lncRNA-155

Deletion of most of the lncRNA sequence

Increased susceptibility to influenza A virus infection

N

HR

[89]

Mdgt

Replacement with a lacZ reporter cassette

Reduced viability and growth

N

HR

[75]

PEAT

Deletion of the entire lncRNA transcribed unit

No apparent phenotype

N/A

Cas9

[29]

Peril

Replacement with a lacZ reporter cassette

Reduction of viability, death shortly after birth as well as reduced body weight

N

HR

[75]

Redrum

Deletion of the lncRNA exon 3

No apparent phenotype

N/A

Cas9 and HR

[30]

Rik-201 and Rik-203

Deletion from the beginning of second exon to the end of the third exon of the lncRNA C130071C03Rik

Abnormal brain development

N

Cas9

[90]

Silc1

Deletion of the lncRNA promoter and exon 1

Delayed regeneration of sensory neurons following injury

N

Cas9

[91]

SRA

Insertion of a lacZ/neo cassette with transcription termination signals before the exon 3

Obesity resistance and improved glucose tolerance in knockout mice fed a high-fat diet

N

HR

[92]

SYISL

Deletion of a 1133-bp genomic region containing most of the SYISL transcript

Increased muscle fiber density, muscle mass, and regeneration

N

Cas9

[93]

Tsix

Deletion of a 3.7-kb CpG-rich domain at the 5′ end of Tsix that included the putative promoter and transcriptional start site

The knockout mice showed normal paternal but impaired maternal transmission. Maternal inheritance is infrequent, with surviving progeny showing intrauterine growth retardation and reduced fertility.

N

HR

[94]

Tsix

Insertion of an IRESβgeo cassette in the second exon to disrupted lncRNA transcripts from both promoters

Inheritance of the disrupted maternal allele resulted in ectopic Xist expression and early embryonic lethality

N

HR

[95]

Tslrn1

Deletion of the entire lncRNA transcribed region

Male knockout mice displayed normal fertility but a significant reduction in spermatozoa.

N

Cas9

[96]

Tsx

Deletion of a 2.1-kb region encompassing the predicted promoter region, exon 1, and 160 bp of intron 1

Male mutant animals have smaller testes and altered behavior with less fear and enhanced short-term memory.

N

HR, FLP and Cre

[97]

Visc-2

Deletion of the entire lncRNA locus

No overt anatomical or behavioral phenotype

N/A

HR

[31]

Air/Airn

Insertion of a polyadenylation cassette to truncate Air to 4% of its length

Mice with the maternally inherited mutant allele were identical to the wild type. Animals with the paternally inherited mutant allele or homozygous mutant mice showed a 15% reduction in birth weight.

N

Y⁷

HR and Cre

[98]

Crnde

Ablation of the whole coding region

Low bone mass phenotype due to impaired osteoblast proliferation and differentiation

N (in vitro rescue only)

Possible⁸

Cas9

[99]

Hand2as/Hand2os1/lncHand2/Uph

Deletion of the exon 1 and/or exon 2

Liver damage and liver regeneration defects

Y

Cas9

[100]

Conditional deletion of the exon 2 in hepatocytes

Severe liver injury, much poorer liver regeneration capacity, and a smaller liver mass

N

Cas9 and Cre

Hand2as/Hand2os1/lncHand2/Uph

Insertion of a triple polyadenylation sequence into the exon 2

Right ventricular hypoplasia and embryonic lethality

N

Y⁹

TALENs

[101]

Hand2as/Hand2os1/lncHand2/Uph

Deletion of the entire lncRNA locus

Septum lesion, heart hypoplasia, and perinatal death

N

Y¹⁰

Cas9

[21]

Deletion of a 2.7-kb DNA sequence that spans exons 4 and 5

Severe contraction defects in adult heart that progressively worsened with increasing age

Deletion of the 5′ promoter and first two exons

No discernable heart phenotypes in either embryos or adults